Kathleen C. Barnes

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.

BackgroundAtopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by a defective skin barrier function. Recent studies have reported mutations of the skin barrier gene encoding filaggrin in a subset of patients with AD.ObjectiveWe investigated whether reduced filaggrin expression was found in patients with AD who were not carriers of known filaggrin mutations and whether filaggrin expression was modulated by the atopic inflammatory response.MethodsFilaggrin expression was measured in skin biopsies and cultured keratinocytes using real-time RT-PCR and immunohistochemistry. Filaggrin loss-of-function mutations were screened in a total of 69 subjects.ResultsCompared with normal skin, filaggrin expression was significantly reduced (P < .05) in acute AD skin, with further reduction seen in acute lesions from 3 European American subjects with AD who were heterozygous for the 2282del4 mutation. This was confirmed by using immunohistochemistry. AD skin is characterized by the overexpression of IL-4 and IL-13. Keratinocytes differentiated in the presence of IL-4 and IL-13 exhibited significantly reduced filaggrin gene expression (0.04 ± 0.01 ng filaggrin/ng glyceraldehyde 3-phosphate dehydrogenase; P < .05) compared with media alone (0.16 ± 0.03).ConclusionPatients with AD have an acquired defect in filaggrin expression that can be modulated by the atopic inflammatory response.Clinical implicationsThe atopic immune response contributes to the skin barrier defect in AD; therefore, neutralization of IL-4 and IL-13 could improve skin barrier integrity. Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by a defective skin barrier function. Recent studies have reported mutations of the skin barrier gene encoding filaggrin in a subset of patients with AD. We investigated whether reduced filaggrin expression was found in patients with AD who were not carriers of known filaggrin mutations and whether filaggrin expression was modulated by the atopic inflammatory response. Filaggrin expression was measured in skin biopsies and cultured keratinocytes using real-time RT-PCR and immunohistochemistry. Filaggrin loss-of-function mutations were screened in a total of 69 subjects. Compared with normal skin, filaggrin expression was significantly reduced (P < .05) in acute AD skin, with further reduction seen in acute lesions from 3 European American subjects with AD who were heterozygous for the 2282del4 mutation. This was confirmed by using immunohistochemistry. AD skin is characterized by the overexpression of IL-4 and IL-13. Keratinocytes differentiated in the presence of IL-4 and IL-13 exhibited significantly reduced filaggrin gene expression (0.04 ± 0.01 ng filaggrin/ng glyceraldehyde 3-phosphate dehydrogenase; P < .05) compared with media alone (0.16 ± 0.03). Patients with AD have an acquired defect in filaggrin expression that can be modulated by the atopic inflammatory response.

Genome-wide association studies (GWAS) have laid the foundation for investigations into the biology of complex traits, drug development and clinical guidelines. However, the majority of discovery efforts are based on data from populations of European ancestry1-3. In light of the differential genetic architecture that is known to exist between populations, bias in representation can exacerbate existing disease and healthcare disparities. Critical variants may be missed if they have a low frequency or are completely absent in European populations, especially as the field shifts its attention towards rare variants, which are more likely to be population-specific4-10. Additionally, effect sizes and their derived risk prediction scores derived in one population may not accurately extrapolate to other populations11,12. Here we demonstrate the value of diverse, multi-ethnic participants in large-scale genomic studies. The Population Architecture using Genomics and Epidemiology (PAGE) study conducted a GWAS of 26 clinical and behavioural phenotypes in 49,839 non-European individuals. Using strategies tailored for analysis of multi-ethnic and admixed populations, we describe a framework for analysing diverse populations, identify 27 novel loci and 38 secondary signals at known loci, as well as replicate 1,444 GWAS catalogue associations across these traits. Our data show evidence of effect-size heterogeneity across ancestries for published GWAS associations, substantial benefits for fine-mapping using diverse cohorts and insights into clinical implications. In the United States-where minority populations have a disproportionately higher burden of chronic conditions13-the lack of representation of diverse populations in genetic research will result in inequitable access to precision medicine for those with the highest burden of disease. We strongly advocate for continued, large genome-wide efforts in diverse populations to maximize genetic discovery and reduce health disparities.

BackgroundAtopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway.ObjectiveWe evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1).MethodsExpression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illumina's BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD.ResultsWe observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with TH2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging SNPs revealed associations with AD in 2 North American populations.ConclusionCollectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1. Atopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. We evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1). Expression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illumina's BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD. We observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with TH2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging SNPs revealed associations with AD in 2 North American populations. Collectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1.

Identifying the ancestry of chromosomal segments of distinct ancestry has a wide range of applications from disease mapping to learning about history. Most methods require the use of unlinked markers; but, using all markers from genome-wide scanning arrays, it should in principle be possible to infer the ancestry of even very small segments with exquisite accuracy. We describe a method, HAPMIX, which employs an explicit population genetic model to perform such local ancestry inference based on fine-scale variation data. We show that HAPMIX outperforms other methods, and we explore its utility for inferring ancestry, learning about ancestral populations, and inferring dates of admixture. We validate the method empirically by applying it to populations that have experienced recent and ancient admixture: 935 African Americans from the United States and 29 Mozabites from North Africa. HAPMIX will be of particular utility for mapping disease genes in recently admixed populations, as its accurate estimates of local ancestry permit admixture and case-control association signals to be combined, enabling more powerful tests of association than with either signal alone.

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).

We examined common variation in asthma risk by conducting a meta-analysis of worldwide asthma genome-wide association studies (23,948 asthma cases, 118,538 controls) of individuals from ethnically diverse populations. We identified five new asthma loci, found two new associations at two known asthma loci, established asthma associations at two loci previously implicated in the comorbidity of asthma plus hay fever, and confirmed nine known loci. Investigation of pleiotropy showed large overlaps in genetic variants with autoimmune and inflammatory diseases. The enrichment in enhancer marks at asthma risk loci, especially in immune cells, suggested a major role of these loci in the regulation of immunologically related mechanisms. The authors perform meta-analysis of GWAS studies for asthma from multiancestral cohorts. They identify five new loci and find that the asthma-associated loci are enriched near enhancer marks in immune cells.

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown1. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer2–4 and coronary heart disease5—this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP)6. Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues. Analysis of 97,691 high-coverage human blood DNA-derived whole-genome sequences enabled simultaneous identification of germline and somatic mutations that predispose individuals to clonal expansion of haematopoietic stem cells, indicating that both inherited and acquired mutations are linked to age-related cancers and coronary heart disease.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by a defective skin barrier function. Recent studies have reported mutations of the skin barrier gene encoding filaggrin in a subset of patients with AD.We investigated whether reduced filaggrin expression was found in patients with AD who were not carriers of known filaggrin mutations and whether filaggrin expression was modulated by the atopic inflammatory response.Filaggrin expression was measured in skin biopsies and cultured keratinocytes using real-time RT-PCR and immunohistochemistry. Filaggrin loss-of-function mutations were screened in a total of 69 subjects.Compared with normal skin, filaggrin expression was significantly reduced (P < .05) in acute AD skin, with further reduction seen in acute lesions from 3 European American subjects with AD who were heterozygous for the 2282del4 mutation. This was confirmed by using immunohistochemistry. AD skin is characterized by the overexpression of IL-4 and IL-13. Keratinocytes differentiated in the presence of IL-4 and IL-13 exhibited significantly reduced filaggrin gene expression (0.04 +/- 0.01 ng filaggrin/ng glyceraldehyde 3-phosphate dehydrogenase; P < .05) compared with media alone (0.16 +/- 0.03).Patients with AD have an acquired defect in filaggrin expression that can be modulated by the atopic inflammatory response.The atopic immune response contributes to the skin barrier defect in AD; therefore, neutralization of IL-4 and IL-13 could improve skin barrier integrity.

Although the pathogenic and genetic basis of acute lung injury (ALI) remains incompletely understood, the identification of novel ALI biomarkers holds promise for unique insights. Expression profiling in animal models of ALI (canine and murine) and human ALI detected significant expression of pre–B-cell colony-enhancing factor (PBEF), a gene not previously associated with lung pathophysiology. These results were validated by real-time polymerase chain reaction and immunohistochemistry studies, with PBEF protein levels significantly increased in both bronchoalveolar lavage fluid and serum of ALI models and in cytokine- or cyclic stretch–activated lung microvascular endothelium. We genotyped two PBEF single-nucleotide polymorphisms (SNPs) in a well characterized sample of white patients with sepsis-associated ALI, patients with severe sepsis, and healthy subjects and observed that carriers of the haplotype GC from SNPs T-1001G and C-1543T had a 7.7-fold higher risk of ALI (95% confidence interval 3.01–19.75, p < 0.001). The T variant from the SNP C-1543T resulted in a significant decrease in the transcription rate (1.8-fold; p < 0.01) by the reporter gene assay. Together, these results strongly indicate that PBEF is a potential novel biomarker in ALI and demonstrate the successful application of robust genomic technologies in the identification of candidate genes in complex lung disease.

Asthma is a complex disease that has genetic and environmental causes. The genetic factors associated with susceptibility to asthma remain largely unknown.We carried out a genomewide association study involving children with asthma. The sample included 793 North American children of European ancestry with persistent asthma who required daily inhaled glucocorticoid therapy and 1988 matched controls (the discovery set). We also tested for genomewide association in an independent cohort of 917 persons of European ancestry who had asthma and 1546 matched controls (the replication set). Finally, we tested for an association between 20 single-nucleotide polymorphisms (SNPs) at chromosome 1q31 and asthma in 1667 North American children of African ancestry who had asthma and 2045 ancestrally matched controls.In our meta-analysis of all samples from persons of European ancestry, we observed an association, with genomewide significance, between asthma and SNPs at the previously reported locus on 17q21 and an additional eight SNPs at a novel locus on 1q31. The SNP most strongly associated with asthma was rs2786098 (P=8.55x10(-9)). We observed replication of the association of asthma with SNP rs2786098 in the independent series of persons of European ancestry (combined P=9.3x10(-11)). The alternative allele of each of the eight SNPs on chromosome 1q31 was strongly associated with asthma in the children of African ancestry (P=1.6x10(-13) for the comparison across all samples). The 1q31 locus contains the 1q31 locus contains DENND1B, a gene expressed by natural killer cells and dendritic cells. DENND1B protein is predicted to interact with the tumor necrosis factor α receptor [corrected].We have identified a locus containing DENND1B on chromosome 1q31.3 that is associated with susceptibility to asthma.

Asthma, a chronic airway disease with known heritability, affects more than 300 million people around the world. A genome-wide association (GWA) study of asthma with 359 cases from the Childhood Asthma Management Program (CAMP) and 846 genetically matched controls from the Illumina ICONdb public resource was performed. The strongest region of association seen was on chromosome 5q12 in PDE4D. The phosphodiesterase 4D, cAMP-specific (phosphodiesterase E3 dunce homolog, Drosophila) gene (PDE4D) is a regulator of airway smooth-muscle contractility, and PDE4 inhibitors have been developed as medications for asthma. Allelic p values for top SNPs in this region were 4.3 x 10(-07) for rs1588265 and 9.7 x 10(-07) for rs1544791. Replications were investigated in ten independent populations with different ethnicities, study designs, and definitions of asthma. In seven white and Hispanic replication populations, two PDE4D SNPs had significant results with p values less than 0.05, and five had results in the same direction as the original population but had p values greater than 0.05. Combined p values for 18,891 white and Hispanic individuals (4,342 cases) in our replication populations were 4.1 x 10(-04) for rs1588265 and 9.2 x 10(-04) for rs1544791. In three black replication populations, which had different linkage disequilibrium patterns than the other populations, original findings were not replicated. Further study of PDE4D variants might lead to improved understanding of the role of PDE4D in asthma pathophysiology and the efficacy of PDE4 inhibitor medications.

Studies in humans and animal models have demonstrated that acute kidney injury (AKI) has a significant effect on the function of extrarenal organs. The combination of AKI and lung dysfunction is associated with 80% mortality; the lung, because of its extensive capillary network, is a prime target for AKI-induced effects. The study presented here tested the hypothesis that AKI leads to a vigorous inflammatory response and produces distinct genomic signatures in the kidney and lung. In a murine model of ischemic AKI, prominent global transcriptomic changes and histologic injury in both kidney and lung tissues were identified. These changes were evident at both early (6 h) and late (36 h) timepoints after 60-min bilateral kidney ischemia and were more prominent than similar timepoints after sham surgery or 30 min of ischemia. The inflammatory transcriptome (109 genes) of both organs changed with marked similarity, including the innate immunity genes Cd14, Socs3, Saa3, Lcn2, and Il1r2. Functional genomic analysis of these genes suggested that IL-10 and IL-6 signaling was involved in the distant effects of local inflammation, and this was supported by increased serum levels of IL-10 and IL-6 after ischemia-reperfusion. In summary, this is the first comprehensive analysis of concomitant inflammation-associated transcriptional changes in the kidney and a remote organ during AKI. Functional genomic analysis identified potential mediators that connect local and systemic inflammation, suggesting that this type of analysis may be a useful discovery tool for novel biomarkers and therapeutic drug development.

To recapitulate, AD is a chronic inflammatory disease of the skin characterized by dysregulation of the adaptive and innate immune response and a heightened IgE-mediated, systemic TH2 response. Extreme TH2 polarization and primary defects in the innate immune response, including epithelial barrier defects, in conjunction with mechanical damage to the epidermis as a consequence of the intense pruritus that is the hallmark feature of AD likely contribute to the more severe sequelae, including chronic bacterial colonization (ie, S aureus infection) and viral dissemination (ie, eczema herpeticum). This scenario can be summarized in Fig 5, whereby the damaged epidermal surface is penetrated by a host of exogenous substances, including allergens, irritants, microbes, pollutants, and even topical drugs. The brick wall–like structure of the SC, which normally creates a barrier that maintains water within the body and prevents the entrance of pathogens and allergens, is further compromised. The EDC, the DNA region in which a large number of genes encoding many of the cornified cell envelope precursor proteins, small proline-rich proteins, members of the S100 family, and intermediate filament-associated protein precursors (ie, profilaggrin) are localized, is an important target of candidate genes associated with barrier dysfunction at the level of the SC. The last line of defense is the stratum granulosum, containing TJs, proteins that constitute the “gate” to the passage of water, ions, and solutes through the paracellular pathway. Systemically, a dysfunctional immune response resulting in an imbalanced innate and adaptive milieu further aggravates the system. Early genome-wide linkage studies, association studies, and high-throughput expression profiling studies have supported the role of skin barrier dysfunction candidate genes in conjunction with innate and adaptive immune response genes. A comprehensive evaluation of all candidate gene studies published to date on AD shows the importance of both sets of genes, and a pathway analysis of the genes studied thus far supports a more thorough approach toward gene-gene and gene-environment interactions.

We used a deeply sequenced dataset of 910 individuals, all of African descent, to construct a set of DNA sequences that is present in these individuals but missing from the reference human genome. We aligned 1.19 trillion reads from the 910 individuals to the reference genome (GRCh38), collected all reads that failed to align, and assembled these reads into contiguous sequences (contigs). We then compared all contigs to one another to identify a set of unique sequences representing regions of the African pan-genome missing from the reference genome. Our analysis revealed 296,485,284 bp in 125,715 distinct contigs present in the populations of African descent, demonstrating that the African pan-genome contains ~10% more DNA than the current human reference genome. Although the functional significance of nearly all of this sequence is unknown, 387 of the novel contigs fall within 315 distinct protein-coding genes, and the rest appear to be intergenic. Assembly of a pan-genome from 910 humans of African descent identifies 296.5 Mb of novel DNA mapping to 125,715 distinct contigs. This African pan-genome contains ~10% more DNA than the current human reference genome.

Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function and is the third leading cause of death globally. Through genome-wide association discovery in 48,943 individuals, selected from extremes of the lung function distribution in UK Biobank, and follow-up in 95,375 individuals, we increased the yield of independent signals for lung function from 54 to 97. A genetic risk score was associated with COPD susceptibility (odds ratio per 1 s.d. of the risk score (∼6 alleles) (95% confidence interval) = 1.24 (1.20-1.27), P = 5.05 × 10-49), and we observed a 3.7-fold difference in COPD risk between individuals in the highest and lowest genetic risk score deciles in UK Biobank. The 97 signals show enrichment in genes for development, elastic fibers and epigenetic regulation pathways. We highlight targets for drugs and compounds in development for COPD and asthma (genes in the inositol phosphate metabolism pathway and CHRM3) and describe targets for potential drug repositioning from other clinical indications.

<h3>Background</h3> A subset of subjects with atopic dermatitis (AD) are susceptible to serious infections with herpes simplex virus, called eczema herpeticum, or vaccina virus, called eczema vaccinatum. <h3>Objective</h3> This National Institute of Allergy and Infectious Diseases–funded multicenter study was performed to establish a database of clinical information and biologic samples on subjects with AD with and without a history of eczema herpeticum (ADEH⁺ and ADEH⁻ subjects, respectively) and healthy control subjects. Careful phenotyping of AD subsets might suggest mechanisms responsible for disseminated viral infections and help identify at-risk individuals. <h3>Methods</h3> We analyzed the data from 901 subjects (ADEH⁺ subjects, n=134; ADEH⁻ subjects, n=419; healthy control subjects, n=348) enrolled between May 11, 2006, and September 16, 2008, at 7 US medical centers. <h3>Results</h3> ADEH⁺ subjects had more severe disease based on scoring systems (Eczema Area and Severity Index and Rajka-Langeland score), body surface area affected, and biomarkers (circulating eosinophil counts and serum IgE, thymus and activation-regulated chemokine, and cutaneous T cell–attracting chemokine) than ADEH⁻ subjects (<i>P</i> < .001). ADEH⁺ subjects were also more likely to have a history of food allergy (69% vs 40%, <i>P</i> < .001) or asthma (64% vs 44%, <i>P</i> < .001) and were more commonly sensitized to many common allergens (<i>P</i> < .001). Cutaneous infections with <i>Staphylococcus aureus</i> or molluscum contagiosum virus were more common in ADEH⁺ subjects (78% and 8%, respectively) than in ADEH⁻ subjects (29% and 2%, respectively; <i>P</i> < .001). <h3>Conclusion</h3> Subjects with AD in whom eczema herpeticum develops have more severe T_H2-polarized disease with greater allergen sensitization and more commonly have a history of food allergy, asthma, or both. They are also much more likely to experience cutaneous infections with <i>S aureus</i> or molluscum contagiosum.

Most genome-wide association and fine-mapping studies to date have been conducted in individuals of European descent, and genetic studies of populations of Hispanic/Latino and African ancestry are limited. In addition, these populations have more complex linkage disequilibrium structure. In order to better define the genetic architecture of these understudied populations, we leveraged >100,000 phased sequences available from deep-coverage whole genome sequencing through the multi-ethnic NHLBI Trans-Omics for Precision Medicine (TOPMed) program to impute genotypes into admixed African and Hispanic/Latino samples with genome-wide genotyping array data. We demonstrated that using TOPMed sequencing data as the imputation reference panel improves genotype imputation quality in these populations, which subsequently enhanced gene-mapping power for complex traits. For rare variants with minor allele frequency (MAF) < 0.5%, we observed a 2.3- to 6.1-fold increase in the number of well-imputed variants, with 11–34% improvement in average imputation quality, compared to the state-of-the-art 1000 Genomes Project Phase 3 and Haplotype Reference Consortium reference panels. Impressively, even for extremely rare variants with minor allele count <10 (including singletons) in the imputation target samples, average information content rescued was >86%. Subsequent association analyses of TOPMed reference panel-imputed genotype data with hematological traits (hemoglobin (HGB), hematocrit (HCT), and white blood cell count (WBC)) in ~21,600 African-ancestry and ~21,700 Hispanic/Latino individuals identified associations with two rare variants in the HBB gene (rs33930165 with higher WBC [p = 8.8x10-15] in African populations, rs11549407 with lower HGB [p = 1.5x10-12] and HCT [p = 8.8x10-10] in Hispanics/Latinos). By comparison, neither variant would have been genome-wide significant if either 1000 Genomes Project Phase 3 or Haplotype Reference Consortium reference panels had been used for imputation. Our findings highlight the utility of the TOPMed imputation reference panel for identification of novel rare variant associations not previously detected in similarly sized genome-wide studies of under-represented African and Hispanic/Latino populations.

Exome sequencing has become a powerful and effective strategy for the discovery of genes underlying Mendelian disorders. However, use of exome sequencing to identify variants associated with complex traits has been more challenging, partly because the sample sizes needed for adequate power may be very large. One strategy to increase efficiency is to sequence individuals who are at both ends of a phenotype distribution (those with extreme phenotypes). Because the frequency of alleles that contribute to the trait are enriched in one or both phenotype extremes, a modest sample size can potentially be used to identify novel candidate genes and/or alleles. As part of the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP), we used an extreme phenotype study design to discover that variants in DCTN4, encoding a dynactin protein, are associated with time to first P. aeruginosa airway infection, chronic P. aeruginosa infection and mucoid P. aeruginosa in individuals with cystic fibrosis.

Analyses of data from genome-wide association studies on unrelated individuals have shown that, for human traits and diseases, approximately one-third to two-thirds of heritability is captured by common SNPs. However, it is not known whether the remaining heritability is due to the imperfect tagging of causal variants by common SNPs, in particular whether the causal variants are rare, or whether it is overestimated due to bias in inference from pedigree data. Here we estimated heritability for height and body mass index (BMI) from whole-genome sequence data on 25,465 unrelated individuals of European ancestry. The estimated heritability was 0.68 (standard error 0.10) for height and 0.30 (standard error 0.10) for body mass index. Low minor allele frequency variants in low linkage disequilibrium (LD) with neighboring variants were enriched for heritability, to a greater extent for protein-altering variants, consistent with negative selection. Our results imply that rare variants, in particular those in regions of low linkage disequilibrium, are a major source of the still missing heritability of complex traits and disease.

Large-scale whole-genome sequencing studies have enabled the analysis of rare variants (RVs) associated with complex phenotypes. Commonly used RV association tests have limited scope to leverage variant functions. We propose STAAR (variant-set test for association using annotation information), a scalable and powerful RV association test method that effectively incorporates both variant categories and multiple complementary annotations using a dynamic weighting scheme. For the latter, we introduce ‘annotation principal components’, multidimensional summaries of in silico variant annotations. STAAR accounts for population structure and relatedness and is scalable for analyzing very large cohort and biobank whole-genome sequencing studies of continuous and dichotomous traits. We applied STAAR to identify RVs associated with four lipid traits in 12,316 discovery and 17,822 replication samples from the Trans-Omics for Precision Medicine Program. We discovered and replicated new RV associations, including disruptive missense RVs of NPC1L1 and an intergenic region near APOC1P1 associated with low-density lipoprotein cholesterol. STAAR is a powerful rare variant association test that incorporates variant functional categories and complementary functional annotations using a dynamic weighting scheme based on annotation principal components. STAAR accounts for population structure and relatedness and is scalable for analyzing large whole-genome sequencing studies.

Patients with atopic dermatitis (AD) are commonly colonized with Staphylococcus aureus (AD S. aureus+), but what differentiates this group from noncolonized AD patients (AD S. aureus–) has not been well studied. To evaluate whether these two groups have unique phenotypic or endotypic features, we performed a multicenter, cross-sectional study enrolling AD S. aureus+ (n = 51) and AD S. aureus– (n = 45) participants defined by the presence or absence of S. aureus by routine culture techniques and nonatopic, noncolonized control individuals (NA S. aureus–) (n = 46). Filaggrin (FLG) genotypes were determined, and disease severity (Eczema Area and Severity Index, Rajka-Langeland Severity Score, Investigator’s Global Assessment score, Numerical Rating Scale, and Dermatology Life Quality Index) was captured. Skin physiology was assessed (transepidermal water loss [TEWL], stratum corneum integrity, hydration, and pH), and serum biomarkers were also measured. We found that AD S. aureus+ patients had more severe disease based on all scoring systems except itch (Numerical Rating Scale), and they had higher levels of type 2 biomarkers (eosinophil count, tIgE, CCL17, and periostin). Additionally, AD S. aureus+ patients had significantly greater allergen sensitization (Phadiatop and tIgE), barrier dysfunction (TEWL and stratum corneum integrity), and serum lactate dehydrogenase (LDH) than both the AD S. aureus– and NA S. aureus– groups. FLG mutations did not associate with S. aureus+ colonization. In conclusion, adult patients with AD who are colonized on their skin with S. aureus have more severe disease, greater type 2 immune deviation, allergen sensitization, barrier disruption, and LDH level elevation than noncolonized patients with AD. Patients with atopic dermatitis (AD) are commonly colonized with Staphylococcus aureus (AD S. aureus+), but what differentiates this group from noncolonized AD patients (AD S. aureus–) has not been well studied. To evaluate whether these two groups have unique phenotypic or endotypic features, we performed a multicenter, cross-sectional study enrolling AD S. aureus+ (n = 51) and AD S. aureus– (n = 45) participants defined by the presence or absence of S. aureus by routine culture techniques and nonatopic, noncolonized control individuals (NA S. aureus–) (n = 46). Filaggrin (FLG) genotypes were determined, and disease severity (Eczema Area and Severity Index, Rajka-Langeland Severity Score, Investigator’s Global Assessment score, Numerical Rating Scale, and Dermatology Life Quality Index) was captured. Skin physiology was assessed (transepidermal water loss [TEWL], stratum corneum integrity, hydration, and pH), and serum biomarkers were also measured. We found that AD S. aureus+ patients had more severe disease based on all scoring systems except itch (Numerical Rating Scale), and they had higher levels of type 2 biomarkers (eosinophil count, tIgE, CCL17, and periostin). Additionally, AD S. aureus+ patients had significantly greater allergen sensitization (Phadiatop and tIgE), barrier dysfunction (TEWL and stratum corneum integrity), and serum lactate dehydrogenase (LDH) than both the AD S. aureus– and NA S. aureus– groups. FLG mutations did not associate with S. aureus+ colonization. In conclusion, adult patients with AD who are colonized on their skin with S. aureus have more severe disease, greater type 2 immune deviation, allergen sensitization, barrier disruption, and LDH level elevation than noncolonized patients with AD.

Tobacco and alcohol use are heritable behaviours associated with 15% and 5.3% of worldwide deaths, respectively, due largely to broad increased risk for disease and injury1-4. These substances are used across the globe, yet genome-wide association studies have focused largely on individuals of European ancestries5. Here we leveraged global genetic diversity across 3.4 million individuals from four major clines of global ancestry (approximately 21% non-European) to power the discovery and fine-mapping of genomic loci associated with tobacco and alcohol use, to inform function of these loci via ancestry-aware transcriptome-wide association studies, and to evaluate the genetic architecture and predictive power of polygenic risk within and across populations. We found that increases in sample size and genetic diversity improved locus identification and fine-mapping resolution, and that a large majority of the 3,823 associated variants (from 2,143 loci) showed consistent effect sizes across ancestry dimensions. However, polygenic risk scores developed in one ancestry performed poorly in others, highlighting the continued need to increase sample sizes of diverse ancestries to realize any potential benefit of polygenic prediction.

Biobanks facilitate genome-wide association studies (GWASs), which have mapped genomic loci across a range of human diseases and traits. However, most biobanks are primarily composed of individuals of European ancestry. We introduce the Global Biobank Meta-analysis Initiative (GBMI)-a collaborative network of 23 biobanks from 4 continents representing more than 2.2 million consented individuals with genetic data linked to electronic health records. GBMI meta-analyzes summary statistics from GWASs generated using harmonized genotypes and phenotypes from member biobanks for 14 exemplar diseases and endpoints. This strategy validates that GWASs conducted in diverse biobanks can be integrated despite heterogeneity in case definitions, recruitment strategies, and baseline characteristics. This collaborative effort improves GWAS power for diseases, benefits understudied diseases, and improves risk prediction while also enabling the nomination of disease genes and drug candidates by incorporating gene and protein expression data and providing insight into the underlying biology of human diseases and traits.

The possibility of voyaging contact between prehistoric Polynesian and Native American populations has long intrigued researchers. Proponents have pointed to the existence of New World crops, such as the sweet potato and bottle gourd, in the Polynesian archaeological record, but nowhere else outside the pre-Columbian Americas1-6, while critics have argued that these botanical dispersals need not have been human mediated7. The Norwegian explorer Thor Heyerdahl controversially suggested that prehistoric South American populations had an important role in the settlement of east Polynesia and particularly of Easter Island (Rapa Nui)2. Several limited molecular genetic studies have reached opposing conclusions, and the possibility continues to be as hotly contested today as it was when first suggested8-12. Here we analyse genome-wide variation in individuals from islands across Polynesia for signs of Native American admixture, analysing 807 individuals from 17 island populations and 15 Pacific coast Native American groups. We find conclusive evidence for prehistoric contact of Polynesian individuals with Native American individuals (around AD 1200) contemporaneous with the settlement of remote Oceania13-15. Our analyses suggest strongly that a single contact event occurred in eastern Polynesia, before the settlement of Rapa Nui, between Polynesian individuals and a Native American group most closely related to the indigenous inhabitants of present-day Colombia.

Fine-mapping to plausible causal variation may be more effective in multi-ancestry cohorts, particularly in the MHC, which has population-specific structure. To enable such studies, we constructed a large (n = 21,546) HLA reference panel spanning five global populations based on whole-genome sequences. Despite population-specific long-range haplotypes, we demonstrated accurate imputation at G-group resolution (94.2%, 93.7%, 97.8% and 93.7% in admixed African (AA), East Asian (EAS), European (EUR) and Latino (LAT) populations). Applying HLA imputation to genome-wide association study data for HIV-1 viral load in three populations (EUR, AA and LAT), we obviated effects of previously reported associations from population-specific HIV studies and discovered a novel association at position 156 in HLA-B. We pinpointed the MHC association to three amino acid positions (97, 67 and 156) marking three consecutive pockets (C, B and D) within the HLA-B peptide-binding groove, explaining 12.9% of trait variance. A high-resolution reference panel based on whole-genome sequencing data enables accurate imputation of HLA alleles across diverse populations and fine-mapping of HLA association signals for HIV-1 host response.

Chronic liver disease is a major public health burden worldwide1. Although different aetiologies and mechanisms of liver injury exist, progression of chronic liver disease follows a common pathway of liver inflammation, injury and fibrosis2. Here we examined the association between clonal haematopoiesis of indeterminate potential (CHIP) and chronic liver disease in 214,563 individuals from 4 independent cohorts with whole-exome sequencing data (Framingham Heart Study, Atherosclerosis Risk in Communities Study, UK Biobank and Mass General Brigham Biobank). CHIP was associated with an increased risk of prevalent and incident chronic liver disease (odds ratio = 2.01, 95% confidence interval (95% CI) [1.46, 2.79]; P < 0.001). Individuals with CHIP were more likely to demonstrate liver inflammation and fibrosis detectable by magnetic resonance imaging compared to those without CHIP (odds ratio = 1.74, 95% CI [1.16, 2.60]; P = 0.007). To assess potential causality, Mendelian randomization analyses showed that genetic predisposition to CHIP was associated with a greater risk of chronic liver disease (odds ratio = 2.37, 95% CI [1.57, 3.6]; P < 0.001). In a dietary model of non-alcoholic steatohepatitis, mice transplanted with Tet2-deficient haematopoietic cells demonstrated more severe liver inflammation and fibrosis. These effects were mediated by the NLRP3 inflammasome and increased levels of expression of downstream inflammatory cytokines in Tet2-deficient macrophages. In summary, clonal haematopoiesis is associated with an elevated risk of liver inflammation and chronic liver disease progression through an aberrant inflammatory response.

Polygenic risk scores (PRSs) have been widely explored in precision medicine. However, few studies have thoroughly investigated their best practices in global populations across different diseases. We here utilized data from Global Biobank Meta-analysis Initiative (GBMI) to explore methodological considerations and PRS performance in 9 different biobanks for 14 disease endpoints. Specifically, we constructed PRSs using pruning and thresholding (P + T) and PRS-continuous shrinkage (CS). For both methods, using a European-based linkage disequilibrium (LD) reference panel resulted in comparable or higher prediction accuracy compared with several other non-European-based panels. PRS-CS overall outperformed the classic P + T method, especially for endpoints with higher SNP-based heritability. Notably, prediction accuracy is heterogeneous across endpoints, biobanks, and ancestries, especially for asthma, which has known variation in disease prevalence across populations. Overall, we provide lessons for PRS construction, evaluation, and interpretation using GBMI resources and highlight the importance of best practices for PRS in the biobank-scale genomics era.

Abstract Most transcriptome-wide association studies (TWASs) so far focus on European ancestry and lack diversity. To overcome this limitation, we aggregated genome-wide association study (GWAS) summary statistics, whole-genome sequences and expression quantitative trait locus (eQTL) data from diverse ancestries. We developed a new approach, TESLA (multi-ancestry integrative study using an optimal linear combination of association statistics), to integrate an eQTL dataset with a multi-ancestry GWAS. By exploiting shared phenotypic effects between ancestries and accommodating potential effect heterogeneities, TESLA improves power over other TWAS methods. When applied to tobacco use phenotypes, TESLA identified 273 new genes, up to 55% more compared with alternative TWAS methods. These hits and subsequent fine mapping using TESLA point to target genes with biological relevance. In silico drug-repurposing analyses highlight several drugs with known efficacy, including dextromethorphan and galantamine, and new drugs such as muscle relaxants that may be repurposed for treating nicotine addiction.

Up-regulation of C-C chemokine expression characterizes allergic inflammation and atopic diseases. A functional mutation in the proximal promoter of the RANTES gene has been identified, which results in a new consensus binding site for the GATA transcription factor family. A higher frequency of this allele was observed in individuals of African descent compared with Caucasian subjects (p < 0.00001). The mutant allele was associated with atopic dermatitis in children of the German Multicenter Allergy Study (MAS-90; p < 0.037), but not with asthma. Transient transfections of the human mast cell line HMC-1 and the T cell line Jurkat with reporter vectors driven by either the mutant or wild-type RANTES promoter showed an up to 8-fold higher constitutive transcriptional activity of the mutant promoter. This is the first report to our knowledge of a functional mutation in a chemokine gene promoter. Our findings suggest that the mutation contributes to the development of atopic dermatitis. Its potential role in other inflammatory and infectious disorders, particularly among individuals of African ancestry, remains to be determined.

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.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.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.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.Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive infection in sepsis.

Loss-of-function null mutations R501X and 2282del4 in the skin barrier gene, filaggrin (FLG), represent the most replicated genetic risk factors for atopic dermatitis (AD). Associations have not been reported in African ancestry populations. Atopic dermatitis eczema herpeticum (ADEH) is a rare but serious complication of AD resulting from disseminated cutaneous herpes simplex virus infections.We aimed to determine whether FLG polymorphisms contribute to ADEH susceptibility.Two common loss-of-function mutations plus 9 FLG single nucleotide polymorphisms were genotyped in 278 European American patients with AD, of whom 112 had ADEH, and 157 nonatopic controls. Replication was performed on 339 African American subjects.Significant associations were observed for both the R501X and 2282del4 mutations and AD among European American subjects (P = 1.46 x 10(-5), 3.87 x 10(-5), respectively), but the frequency of the R501X mutation was 3 times higher (25% vs 9%) for ADEH than for AD without eczema herpeticum (EH) (odds ratio [OR], 3.4; 1.7-6.8; P = .0002). Associations with ADEH were stronger with the combined null mutations (OR, 10.1; 4.7-22.1; P = 1.99 x 10(-11)). Associations with the R501X mutation were replicated in the African American population; the null mutation was absent among healthy African American subjects, but present among patients with AD (3.2%; P = .035) and common among patients with ADEH (9.4%; P = .0049). However, the 2282del4 mutation was absent among African American patients with ADEH and rare (<1%) among healthy individuals.The R501X mutation in the gene encoding filaggrin, one of the strongest genetic predictors of AD, confers an even greater risk for ADEH in both European and African ancestry populations, suggesting a role for defective skin barrier in this devastating condition.

Interpatient variability in montelukast response may be related to variation in leukotriene pathway candidate genes.To determine associations between polymorphisms in leukotriene pathway candidate genes with outcomes in patients with asthma receiving montelukast for 6 mo who participated in a clinical trial.Polymorphisms were typed using Sequenom matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass array spectrometry and published methods; haplotypes were imputed using single nucleotide polymorphism-expectation maximization (SNP-EM). Analysis of variance and logistic regression models were used to test for changes in outcomes by genotype. In addition, chi(2) and likelihood ratio tests were used to test for differences between groups. Case-control comparisons were analyzed using the SNP-EM Omnibus likelihood ratio test.Outcomes were asthma exacerbation rate and changes in FEV(1) compared with baseline.DNA was collected from 252 participants: 69% were white, 26% were African American. Twenty-eight SNPs in the ALOX5, LTA4H, LTC4S, MRP1, and cysLT1R genes, and an ALOX5 repeat polymorphism were successfully typed. There were racial disparities in allele frequencies in 17 SNPs and in the repeat polymorphism. Association analyses were performed in 61 whites. Associations were found between genotypes of SNPs in the ALOX5 (rs2115819) and MRP1 (rs119774) genes and changes in FEV(1) (p < 0.05), and between two SNPs in LTC4S (rs730012) and in LTA4H (rs2660845) genes for exacerbation rates. Mutant ALOX5 repeat polymorphism was associated with decreased exacerbation rates. There was strong linkage disequilibrium between ALOX5 SNPs. Associations between ALOX5 haplotypes and risk of exacerbations were found.Genetic variation in leukotriene pathway candidate genes contributes to variability in montelukast response.

The genomewide screen to search for asthma-susceptibility loci, in the Collaborative Study on the Genetics of Asthma (CSGA), has been conducted in two stages and includes 266 families (199 nuclear and 67 extended pedigrees) from three U.S. populations: African American, European American, and Hispanic. Evidence for linkage with the asthma phenotype was observed for multiple chromosomal regions, through use of several analytical approaches that facilitated the identification of multiple disease loci. Ethnicity-specific analyses, which allowed for different frequencies of asthma-susceptibility genes in each ethnic population, provided the strongest evidence for linkage at 6p21 in the European American population, at 11q21 in the African American population, and at 1p32 in the Hispanic population. Both the conditional analysis and the affected-sib-pair two-locus analysis provided further evidence for linkage, at 5q31, 8p23, 12q22, and 15q13. Several of these regions have been observed in other genomewide screens and linkage or association studies, for asthma and related phenotypes. These results were used to develop a conceptual model to delineate asthma-susceptibility loci and their genetic interactions, which provides a promising basis for initiation of fine-mapping studies and, ultimately, for gene identification.

To identify genes potentially relevant in atopic asthma, we analyzed markers in chromosome 12q15–q24.1 for linkage to asthma and total serum IgE concentration. Sib-pair analyses of 10 markers in 345 full- and 219 half-sib pairs from 29 multiplex Afro-Caribbean families provided evidence for linkage to this region for both asthma and total serum IgE. Certain alleles at these loci showed significant evidence of transmission disequilibrium with both asthma and high IgE. Using 6 of these markers and 11 additional markers, evidence for linkage of total IgE to 12q was also found in 12 Caucasian Amish kindreds (24 nuclear families) by both sib-pair and transmission disequilibrium analyses. These findings suggest that the 12q15–q24.1 region may contain a gene(s) controlling asthma and the associated “high total IgE” trait.

Thymic stromal lymphopoietin (TSLP) triggers dendritic cell--mediated T helper (Th) 2 inflammatory responses. A single-nucleotide polymorphism (SNP), rs3806933, in the promoter region of the TSLP gene creates a binding site for the transcription factor activating protein (AP)-1. The variant enhances AP-1 binding to the regulatory element, and increases the promoter--reporter activity of TSLP in response to polyinosinic-polycytidylic acid (poly[I:C]) stimulation in normal human bronchial epithelium (NHBE). We investigated whether polymorphisms including the SNP rs3806933 could affect the susceptibility to and clinical phenotypes of bronchial asthma. We selected three representative (i.e., Tag) SNPs and conducted association studies of the TSLP gene, using two independent populations (639 patients with childhood atopic asthma and 838 control subjects, and 641 patients with adult asthma and 376 control subjects, respectively). We further examined the effects of corticosteroids and a long-acting β(2)-agonist (salmeterol) on the expression levels of the TSLP gene in response to poly(I:C) in NHBE. We found that the promoter polymorphisms rs3806933 and rs2289276 were significantly associated with disease susceptibility in both childhood atopic and adult asthma. The functional SNP rs3806933 was associated with asthma (meta-analysis, P = 0.000056; odds ratio, 1.29; 95% confidence interval, 1.14-1.47). A genotype of rs2289278 was correlated with pulmonary function. Moreover, the induction of TSLP mRNA and protein expression induced by poly(I:C) in NHBE was synergistically impaired by a corticosteroid and salmeterol. TSLP variants are significantly associated with bronchial asthma and pulmonary function. Thus, TSLP may serve as a therapeutic target molecule for combination therapy.

BackgroundAsthma is a complex disease characterized by striking ethnic disparities not explained entirely by environmental, social, cultural, or economic factors. Of the limited genetic studies performed on populations of African descent, notable differences in susceptibility allele frequencies have been observed.ObjectivesWe sought to test the hypothesis that some genes might contribute to the profound disparities in asthma.MethodsWe performed a genome-wide association study in 2 independent populations of African ancestry (935 African American asthmatic cases and control subjects from the Baltimore–Washington, DC, area and 929 African Caribbean asthmatic subjects and their family members from Barbados) to identify single-nucleotide polymorphisms (SNPs) associated with asthma.ResultsA meta-analysis combining these 2 African-ancestry populations yielded 3 SNPs with a combined P value of less than 10−5 in genes of potential biologic relevance to asthma and allergic disease: rs10515807, mapping to the α-1B-adrenergic receptor (ADRA1B) gene on chromosome 5q33 (3.57 × 10−6); rs6052761, mapping to the prion-related protein (PRNP) gene on chromosome 20pter-p12 (2.27 × 10−6); and rs1435879, mapping to the dipeptidyl peptidase 10 (DPP10) gene on chromosome 2q12.3-q14.2. The generalizability of these findings was tested in family and case-control panels of United Kingdom and German origin, respectively, but none of the associations observed in the African groups were replicated in these European studies. Evidence for association was also examined in 4 additional case-control studies of African Americans; however, none of the SNPs implicated in the discovery population were replicated.ConclusionsThis study illustrates the complexity of identifying true associations for a complex and heterogeneous disease, such as asthma, in admixed populations, especially populations of African descent. Asthma is a complex disease characterized by striking ethnic disparities not explained entirely by environmental, social, cultural, or economic factors. Of the limited genetic studies performed on populations of African descent, notable differences in susceptibility allele frequencies have been observed. We sought to test the hypothesis that some genes might contribute to the profound disparities in asthma. We performed a genome-wide association study in 2 independent populations of African ancestry (935 African American asthmatic cases and control subjects from the Baltimore–Washington, DC, area and 929 African Caribbean asthmatic subjects and their family members from Barbados) to identify single-nucleotide polymorphisms (SNPs) associated with asthma. A meta-analysis combining these 2 African-ancestry populations yielded 3 SNPs with a combined P value of less than 10−5 in genes of potential biologic relevance to asthma and allergic disease: rs10515807, mapping to the α-1B-adrenergic receptor (ADRA1B) gene on chromosome 5q33 (3.57 × 10−6); rs6052761, mapping to the prion-related protein (PRNP) gene on chromosome 20pter-p12 (2.27 × 10−6); and rs1435879, mapping to the dipeptidyl peptidase 10 (DPP10) gene on chromosome 2q12.3-q14.2. The generalizability of these findings was tested in family and case-control panels of United Kingdom and German origin, respectively, but none of the associations observed in the African groups were replicated in these European studies. Evidence for association was also examined in 4 additional case-control studies of African Americans; however, none of the SNPs implicated in the discovery population were replicated. This study illustrates the complexity of identifying true associations for a complex and heterogeneous disease, such as asthma, in admixed populations, especially populations of African descent.

Mutations in the essential telomerase genes TERT and TR cause familial pulmonary fibrosis; however, in telomerase-null mice, short telomeres predispose to emphysema after chronic cigarette smoke exposure. Here, we tested whether telomerase mutations are a risk factor for human emphysema by examining their frequency in smokers with chronic obstructive pulmonary disease (COPD). Across two independent cohorts, we found 3 of 292 severe COPD cases carried deleterious mutations in TERT (1%). This prevalence is comparable to the frequency of alpha-1 antitrypsin deficiency documented in this population. The TERT mutations compromised telomerase catalytic activity, and mutation carriers had short telomeres. Telomerase mutation carriers with emphysema were predominantly female and had an increased incidence of pneumothorax. In families, emphysema showed an autosomal dominant inheritance pattern, along with pulmonary fibrosis and other telomere syndrome features, but manifested only in smokers. Our findings identify germline mutations in telomerase as a Mendelian risk factor for COPD susceptibility that clusters in autosomal dominant families with telomere-mediated disease including pulmonary fibrosis.

To the Editor:Atopic dermatitis (AD) is a chronic skin disease affecting up to 20% of children in industrialized countries. A rare but serious complication of AD is eczema herpeticum (EH). We recently reported that subjects with AD with EH (ADEH) have more severe TH2-polarized disease with greater allergen sensitization and more commonly have a history of food allergy, asthma, or both.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Only approximately 5% of patients with AD with herpes simplex virus (HSV) seropositivity (eg, evidence of exposure) will have EH.2Tay Y.K. Khoo B.P. Goh C.L. The epidemiology of atopic dermatitis at a tertiary referral skin center in Singapore.Asian Pac J Allergy Immunol. 1999; 17: 137-141PubMed Google Scholar This observation, coupled with the evidence that susceptibility to EH can be familial and that most subjects report recurrent EH episodes, suggests that genetic susceptibility might be important.Thymic stromal lymphopoietin (TSLP) is an IL-7–like cytokine that triggers dendritic cells to induce differentiation of naive T cells into TH2 cells and is implicated in the pathogenesis of allergic diseases.3Liu Y.J. Soumelis V. Watanabe N. Ito T. Wang Y.H. Malefyt Rde W. et al.TSLP: an epithelial cell cytokine that regulates T cell differentiation by conditioning dendritic cell maturation.Annu Rev Immunol. 2007; 25: 193-219Crossref PubMed Scopus (527) Google ScholarTSLP exerts its biological activities by binding to a heterodimeric receptor consisting of the IL-7 receptor α chain (IL-7Rα) and the TSLP receptor chain (TSLPR) to initiate signal transducer and activator of transcription 3 and 5 phosphorylation.4Pandey A. Ozaki K. Baumann H. Levin S.D. Puel A. Farr A.G. et al.Cloning of a receptor subunit required for signaling by thymic stromal lymphopoietin.Nat Immunol. 2000; 1: 59-64Crossref PubMed Scopus (334) Google Scholar Recent studies have demonstrated that polymorphisms of the TSLP gene appear to contribute to TH2-polarized immunity through higher TSLP production by bronchial epithelial cells in response to viral respiratory tract infections.5Harada M. Hirota T. Jodo A.I. Doi S. Kameda M. Fujita K. et al.Functional analysis of the thymic stromal lymphopoietin variants in human bronchial epithelial cells.Am J Respir Cell Mol Biol. 2009; 40: 368-374Crossref PubMed Scopus (127) Google Scholar In this study we hypothesized that variants in TSLP and its receptors were associated with the risk of AD, ADEH, and related subphenotypes. To test the hypothesis, we conducted genetic association studies in 2 independent and racially diverse groups of patients participating in the multicenter Atopic Dermatitis Vaccinia Network (ADVN).1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Detailed information on the participants in the ADVN has been previously described.6Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124 (e1-7): 507-513Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Local institutional review boards and clinics approved the study, and written informed consent was obtained from all study participants.A total of 29 single nucleotide polymorphisms (SNPs) were selected from TSLP, IL7R, and TSLPR (15, 11, and 3, respectively) for genotyping. Of these, there were 23 tagging SNPs, one recently reported TSLP functional SNP (rs3806933, −847C/T), 2 SNPs (rs1898671 and rs10062929) within the initially identified region of TSLP, and 3 newly validated TSLPR dbSNPs. Details for each SNP and minor allele frequencies (MAFs) are presented in Table E1 in this article's Online Repository at www.jacionline.org. Of these, 20 SNPs were genotyped by using a custom-designed Illumina (San Diego, Calif) oligonucleotide pool assay (OPA) for the BeadXpress Reader System, and 9 SNPs were genotyped with ABI TaqMan system (Applied Biosystems, Foster City, Calif). Quality controls were performed as described previously.6Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124 (e1-7): 507-513Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar The Cochran-Armitage trend test was used to test for association between each SNP and disease status by using PLINK.7Purcell S. Neale B. Todd-Brown K. Thomas L. Ferreira M.A. Bender D. et al.PLINK: a tool set for whole-genome association and population-based linkage analyses.Am J Hum Genet. 2007; 81: 559-575Abstract Full Text Full Text PDF PubMed Scopus (18978) Google Scholar A linear regression analysis was performed to test for associations between individual genetic markers and log-adjusted total serum IgE (tIgE) concentrations and log-adjusted Eczema Area and Severity Index (EASI) scores, and a multiple logistic regression model was used to test for SNP-SNP interactions among cases only between TSLP and its receptors by using SAS version 9.1 software (SAS Institute, Inc, Cary, NC).As shown in Fig 1, A, and Table I, among the primary European American group, a significant association was observed for TSLP SNP rs11466749 and AD (P = .028). Significant associations were also observed for ADEH and 2 additional TSLP SNPs (rs1898671, P = .001; rs2416259, P = .021) and 4 IL7R SNPs (rs12516866, P = .044; rs10213865, P = .027; rs1389832; P = .011; rs10058453, P = .022). When replication was sought in the smaller African American sample, modest associations were observed for AD and 2 TSLP SNPs (rs10043985, P = .034; rs2289276, P = .018) and 2 IL7R SNPs (rs12516866, P = .033; rs1053496, P = .011). The number of patients with ADEH in the African American sample was too small to perform meaningful tests for association. To test for association with disease severity, we performed a linear regression analysis using an additive model. Results are summarized in Fig 1, B, and Table E2 in this article's Online Repository at www.jacionline.org. Associations with tIgE concentrations were observed for 6 TSLP SNPs, including 3 SNPs in the European American sample (rs10062929, P = .031; rs11466750, P = .048; rs2416259, P = .034), 3 SNPs in the African American sample (rs991035, P = .012; rs17551370, P = .023; rs11466749, P = .015), and 1 IL7R SNP (rs7737000, P = .024) in the African American sample. Two TSLP SNPs associated with tIgE concentrations were also significantly associated with the EASI score, including rs2416259 in the European American sample (P = .011) and rs991035 in the African American sample (P = .013). To identify functional mutations for TSLPR, we sequenced the TSLPR coding regions and validated 3 dbSNPs with allele frequencies of greater than 10%: rs36139698 (P196L), rs36177645 (X201W), and rs36133495 (A238V). All 3 SNPs were modestly associated with AD in the European American sample (rs36139698, P = .026; rs36177645, P = .039; rs36133495, P = .041; Table I), but this was not replicated in the African American sample. When patients with AD were limited to those of male sex, the most significant association was observed for the SNP rs36133495 (P = .0005, data not shown). Given the biological relationship between TSLP and its receptors, we tested for epistatic effects (ie, gene-gene interaction) and observed significant interactive effects for AD and ADEH between TSLP and IL7R and TSLPR (see Table E3 in this article's Online Repository at www.jacionline.org). The strongest interaction was observed for AD between TSLP SNP rs2289276 and IL7R SNP rs12516866 (odds ratio, 1.86; P = .007) among European American subjects and between TSLP SNP rs1898671 and IL7R SNP rs10213865 (odds ratio, 5.12; P = .005) among African American subjects.Table IAssociation tests between TSLP, IL7R, and TSLPR SNPs and AD and ADEH in European American (n = 444) and African American (n = 339) subjectsGeneMarkerBase changeRoleEuropean American subjectsAfrican American subjectsPatients with AD∗ADEH+ subjects plus ADEH− subjects. vs healthy subjectsADEH+ subjects vs ADEH− subjectsPatients with AD∗ADEH+ subjects plus ADEH− subjects. vs healthy subjectsOR (95% CI)P valueOR (95% CI)P valueOR (95% CI)P valueTSLPrs10043985A/CPromoterNANANANA0.5 (0.3-1.0).034rs2289276C/T5′-UTR1.3 (0.8-1.9).2701.2 (0.7-2.1).4801.8 (1.1-3.1).018rs1898671C/TIntron0.9 (0.7-1.3).6190.6 (0.4-0.9).0011.3 (0.8-2.1).306rs11466749A/G3′-UTR0.6 (0.4-1.0).0280.7 (0.3-1.3).2130.7 (0.5-1.3).321rs2416259T/CDownstream0.8 (0.5-1.3).3562.0 (1.1-3.6).0211.0 (0.4-2.1).906IL7Rrs12516866G/TPromoter0.7 (0.4-1.1).1080.6 (0.3-1.0).0440.8 (0.5-1.2).033rs10213865A/CIntron0.7 (0.5-1.1).1140.6 (0.3-1.0).0270.7 (0.4-1.1).119rs1389832C/TIntron1.1 (0.7-1.7).5082.0 (1.1-3.7).0111.2 (0.7-2.0).464rs1053496C/TDownstream1.1 (0.7-1.7).5611.2 (0.7-2.1).4531.8 (1.1-2.9).011rs10058453T/CDownstream0.9 (0.6-1.4).6741.9 (1.1-3.4).0221.2 (0.7-1.8).555TSLPRrs36139698C/T (P196L)Exon0.6 (0.4-1.0).0260.9 (0.5-1.5).0641.1 (0.7-1.8).584rs36177645A/G (X210W)Exon1.5 (1.0-2.3).0391.6 (0.9-2.7).0961.0 (0.6-1.6).916rs36133495C/T (A238V)Exon1.6 (1.0-2.7).0411.2 (0.6-2.5).5530.9 (0.5-1.4).547Odds ratios (ORs) and P values were calculated under the dominant model. Boldfaced odds ratios and P values are statistically significant.UTR, Untranslated region.∗ ADEH+ subjects plus ADEH− subjects. Open table in a new tab In this study we observed significant associations for TSLP and IL7R tagging SNPs and AD and ADEH among a European American sample, with replication of associations between TSLP and IL7R SNPs in an independent African American sample. A SNP (rs1898671) in TSLP showed the strongest association with a lower risk of ADEH among European American subjects (P = .001), which remained significant after a Bonferroni correction for multiple testing (P = .015). Although the function of this variant is unknown, 2 SNPs (rs3806933 and rs2289276) in the promoter region of TSLP are in strong linkage disequilibrium (LD) with rs1898671, and all 3 markers are localized in a single LD block (Fig 1, C). Elsewhere, these markers have been associated with an increase in promoter activity and enhanced activator protein 1 (AP-1) binding to the regulatory element of TSLP.5Harada M. Hirota T. Jodo A.I. Doi S. Kameda M. Fujita K. et al.Functional analysis of the thymic stromal lymphopoietin variants in human bronchial epithelial cells.Am J Respir Cell Mol Biol. 2009; 40: 368-374Crossref PubMed Scopus (127) Google Scholar SNP rs2416259 was also associated with risk of ADEH, as well as associated phenotypes, high tIgE concentrations, and EASI scores. Of interest, SNP rs2289276, an SNP previously associated with specific IgE levels to cockroach antigen and total IgE concentration,8Hunninghake G.M. Lasky-Su J. Soto-Quiros M.E. Avila L. Liang C. Lake S.L. et al.Sex-stratified linkage analysis identifies a female-specific locus for IgE to cockroach in Costa Ricans.Am J Respir Crit Care Med. 2008; 177: 830-836Crossref PubMed Scopus (62) Google Scholar was associated with AD in our study. Three validated coding dbSNPs in TSLPR were significantly associated with AD in European American subjects, one of which is a nonsense SNP (rs36177645 X201W), which might lead to premature termination of peptides; investigation into the effects on gene expression are underway. Potential epistatic effects between SNPs in TSLP and its 2 receptors, IL7R and TSLPR, were observed for both AD and ADEH. Associations for SNP-SNP interactions were stronger than the single-marker associations.A gene-for-gene replication of associations was observed in the African American sample for the traits analyzed (AD, tIgE concentration, and EASI score) and TSLP and IL7R SNPs. Because of the smaller African American ADEH sample, it was not possible to test for replication to the ADEH trait. However, the SNPs significantly associated with AD, tIgE concentration, and EASI score in the African American subjects were not for the same SNPs as those associated with these 3 traits in the European American subjects. Failure to observe SNP-for-SNP replication in ethnically diverse populations is not uncommon,9Cui Y. Kang G. Sun K. Qian M. Romero R. Fu W. Gene-centric genomewide association study via entropy.Genetics. 2008; 179: 637-650Crossref PubMed Scopus (33) Google Scholar and potential reasons include variation in allele frequencies, genetic structure, and heterogeneity of the phenotype and environment. Heterogeneity of the phenotype is unlikely in this case because all cases were characterized by using identical protocols at multiple centers.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Previously, we evaluated the potential effect of admixture on the case-control design using ancestry informative markers,6Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124 (e1-7): 507-513Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar but we observed no significant population stratification in this sample. However, nearly half (44.8%) of the SNPs genotyped in the current study had substantially different MAFs between the 2 populations, highlighting considerable genetic heterogeneity between the 2 ethnic groups, and the LD structure differed considerably in these 2 samples (data not shown). Very few of the markers genotyped in this study are likely to be causal but rather are tagging SNPs in LD with an unknown disease-causing variant or variants. That said, it is well accepted that African Americans are more genetically diverse than populations of European ancestry and have shorter LD blocks, and it is therefore not surprising that different sets of tagging SNPs might be associated with the trait of interest compared with European American subjects. We contend that by capturing all of the potential risk-conferring variants, a gene-based approach, rather than an SNP-for-SNP approach, might provide evidence for genetic analysis at the functional level.10Neale B.M. Sham P.C. The future of association studies: gene-based analysis and replication.Am J Hum Genet. 2004; 75: 353-362Abstract Full Text Full Text PDF PubMed Scopus (520) Google ScholarOther confounders could influence the observed associations in this study, both between case groups and across ethnic groups. Most cases of ADEH are caused by HSV-1, despite the fact that HSV-1 seropositivity is high in the general population.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar In the ADVN study 93% of the ADEH group was seropositive for HSV-1 compared with seropositivity of just over 50% for patients with AD without ADEH (ADEH negative, 52%) and control subjects (54%).1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar To test for the possibility that the associations observed between TSLP, IL7R, and TSLPR were for manifestations of an HSV exacerbation in an AD population rather than the disease markers, we investigated the association between HSV infection as determined by both HSV-1 and HSV-2 positivity and the associated SNPs among control subjects, none of which showed enhanced associations compared with the associations with ADEH (data not shown). Because there were no statistical differences in HSV infection between ADEH-negative and control subjects,1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar we also tested for association between the associated SNPs and ADEH-negative status and found that the associations with AD remained (data not shown). These results suggest that the associations between AD and ADEH and TSLP, IL7R, and TSLPR are independent of HSV infection, although a more robustly powered sample is needed to verify these findings. We acknowledge that this study was marginally powered overall, largely because of the rarity of the trait of interest, ADEH, which occurs in less than 5% of subjects with AD. Given the candidate gene nature of this study and the strong biological justification for interrogating the role of TSLP variants and variants in its receptors in the risk of ADEH, AD, or both, the Bonferroni correction for multiple tests is likely too conservative. Nevertheless, one of the most compelling associations was between ADEH and a SNP (rs1898671) in TSLP that is in strong LD with a functional variant previously associated with enhanced AP-1 binding to the regulatory element of TSLP,5Harada M. Hirota T. Jodo A.I. Doi S. Kameda M. Fujita K. et al.Functional analysis of the thymic stromal lymphopoietin variants in human bronchial epithelial cells.Am J Respir Cell Mol Biol. 2009; 40: 368-374Crossref PubMed Scopus (127) Google Scholar an association that remained significant after correcting for multiple testing.In summary, we demonstrate evidence of association between markers in TSLP and its receptors, IL7R and TSLPR, and the risk of AD and its most serious complication, ADEH, in a multicenter case-control study. Our findings suggest that TSLP might be an important candidate for AD, AD severity, and ADEH. This is the first study to implicate TSLP as a potential casual gene for ADEH. A clearer understanding of these risk factors might improve our ability to identify patients at greatest risk for ADEH, which might ultimately lead to early intervention and improved surveillance in this vulnerable population. To the Editor: Atopic dermatitis (AD) is a chronic skin disease affecting up to 20% of children in industrialized countries. A rare but serious complication of AD is eczema herpeticum (EH). We recently reported that subjects with AD with EH (ADEH) have more severe TH2-polarized disease with greater allergen sensitization and more commonly have a history of food allergy, asthma, or both.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Only approximately 5% of patients with AD with herpes simplex virus (HSV) seropositivity (eg, evidence of exposure) will have EH.2Tay Y.K. Khoo B.P. Goh C.L. The epidemiology of atopic dermatitis at a tertiary referral skin center in Singapore.Asian Pac J Allergy Immunol. 1999; 17: 137-141PubMed Google Scholar This observation, coupled with the evidence that susceptibility to EH can be familial and that most subjects report recurrent EH episodes, suggests that genetic susceptibility might be important. Thymic stromal lymphopoietin (TSLP) is an IL-7–like cytokine that triggers dendritic cells to induce differentiation of naive T cells into TH2 cells and is implicated in the pathogenesis of allergic diseases.3Liu Y.J. Soumelis V. Watanabe N. Ito T. Wang Y.H. Malefyt Rde W. et al.TSLP: an epithelial cell cytokine that regulates T cell differentiation by conditioning dendritic cell maturation.Annu Rev Immunol. 2007; 25: 193-219Crossref PubMed Scopus (527) Google ScholarTSLP exerts its biological activities by binding to a heterodimeric receptor consisting of the IL-7 receptor α chain (IL-7Rα) and the TSLP receptor chain (TSLPR) to initiate signal transducer and activator of transcription 3 and 5 phosphorylation.4Pandey A. Ozaki K. Baumann H. Levin S.D. Puel A. Farr A.G. et al.Cloning of a receptor subunit required for signaling by thymic stromal lymphopoietin.Nat Immunol. 2000; 1: 59-64Crossref PubMed Scopus (334) Google Scholar Recent studies have demonstrated that polymorphisms of the TSLP gene appear to contribute to TH2-polarized immunity through higher TSLP production by bronchial epithelial cells in response to viral respiratory tract infections.5Harada M. Hirota T. Jodo A.I. Doi S. Kameda M. Fujita K. et al.Functional analysis of the thymic stromal lymphopoietin variants in human bronchial epithelial cells.Am J Respir Cell Mol Biol. 2009; 40: 368-374Crossref PubMed Scopus (127) Google Scholar In this study we hypothesized that variants in TSLP and its receptors were associated with the risk of AD, ADEH, and related subphenotypes. To test the hypothesis, we conducted genetic association studies in 2 independent and racially diverse groups of patients participating in the multicenter Atopic Dermatitis Vaccinia Network (ADVN).1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar Detailed information on the participants in the ADVN has been previously described.6Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124 (e1-7): 507-513Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Local institutional review boards and clinics approved the study, and written informed consent was obtained from all study participants. A total of 29 single nucleotide polymorphisms (SNPs) were selected from TSLP, IL7R, and TSLPR (15, 11, and 3, respectively) for genotyping. Of these, there were 23 tagging SNPs, one recently reported TSLP functional SNP (rs3806933, −847C/T), 2 SNPs (rs1898671 and rs10062929) within the initially identified region of TSLP, and 3 newly validated TSLPR dbSNPs. Details for each SNP and minor allele frequencies (MAFs) are presented in Table E1 in this article's Online Repository at www.jacionline.org. Of these, 20 SNPs were genotyped by using a custom-designed Illumina (San Diego, Calif) oligonucleotide pool assay (OPA) for the BeadXpress Reader System, and 9 SNPs were genotyped with ABI TaqMan system (Applied Biosystems, Foster City, Calif). Quality controls were performed as described previously.6Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124 (e1-7): 507-513Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar The Cochran-Armitage trend test was used to test for association between each SNP and disease status by using PLINK.7Purcell S. Neale B. Todd-Brown K. Thomas L. Ferreira M.A. Bender D. et al.PLINK: a tool set for whole-genome association and population-based linkage analyses.Am J Hum Genet. 2007; 81: 559-575Abstract Full Text Full Text PDF PubMed Scopus (18978) Google Scholar A linear regression analysis was performed to test for associations between individual genetic markers and log-adjusted total serum IgE (tIgE) concentrations and log-adjusted Eczema Area and Severity Index (EASI) scores, and a multiple logistic regression model was used to test for SNP-SNP interactions among cases only between TSLP and its receptors by using SAS version 9.1 software (SAS Institute, Inc, Cary, NC). As shown in Fig 1, A, and Table I, among the primary European American group, a significant association was observed for TSLP SNP rs11466749 and AD (P = .028). Significant associations were also observed for ADEH and 2 additional TSLP SNPs (rs1898671, P = .001; rs2416259, P = .021) and 4 IL7R SNPs (rs12516866, P = .044; rs10213865, P = .027; rs1389832; P = .011; rs10058453, P = .022). When replication was sought in the smaller African American sample, modest associations were observed for AD and 2 TSLP SNPs (rs10043985, P = .034; rs2289276, P = .018) and 2 IL7R SNPs (rs12516866, P = .033; rs1053496, P = .011). The number of patients with ADEH in the African American sample was too small to perform meaningful tests for association. To test for association with disease severity, we performed a linear regression analysis using an additive model. Results are summarized in Fig 1, B, and Table E2 in this article's Online Repository at www.jacionline.org. Associations with tIgE concentrations were observed for 6 TSLP SNPs, including 3 SNPs in the European American sample (rs10062929, P = .031; rs11466750, P = .048; rs2416259, P = .034), 3 SNPs in the African American sample (rs991035, P = .012; rs17551370, P = .023; rs11466749, P = .015), and 1 IL7R SNP (rs7737000, P = .024) in the African American sample. Two TSLP SNPs associated with tIgE concentrations were also significantly associated with the EASI score, including rs2416259 in the European American sample (P = .011) and rs991035 in the African American sample (P = .013). To identify functional mutations for TSLPR, we sequenced the TSLPR coding regions and validated 3 dbSNPs with allele frequencies of greater than 10%: rs36139698 (P196L), rs36177645 (X201W), and rs36133495 (A238V). All 3 SNPs were modestly associated with AD in the European American sample (rs36139698, P = .026; rs36177645, P = .039; rs36133495, P = .041; Table I), but this was not replicated in the African American sample. When patients with AD were limited to those of male sex, the most significant association was observed for the SNP rs36133495 (P = .0005, data not shown). Given the biological relationship between TSLP and its receptors, we tested for epistatic effects (ie, gene-gene interaction) and observed significant interactive effects for AD and ADEH between TSLP and IL7R and TSLPR (see Table E3 in this article's Online Repository at www.jacionline.org). The strongest interaction was observed for AD between TSLP SNP rs2289276 and IL7R SNP rs12516866 (odds ratio, 1.86; P = .007) among European American subjects and between TSLP SNP rs1898671 and IL7R SNP rs10213865 (odds ratio, 5.12; P = .005) among African American subjects. Odds ratios (ORs) and P values were calculated under the dominant model. Boldfaced odds ratios and P values are statistically significant. UTR, Untranslated region. In this study we observed significant associations for TSLP and IL7R tagging SNPs and AD and ADEH among a European American sample, with replication of associations between TSLP and IL7R SNPs in an independent African American sample. A SNP (rs1898671) in TSLP showed the strongest association with a lower risk of ADEH among European American subjects (P = .001), which remained significant after a Bonferroni correction for multiple testing (P = .015). Although the function of this variant is unknown, 2 SNPs (rs3806933 and rs2289276) in the promoter region of TSLP are in strong linkage disequilibrium (LD) with rs1898671, and all 3 markers are localized in a single LD block (Fig 1, C). Elsewhere, these markers have been associated with an increase in promoter activity and enhanced activator protein 1 (AP-1) binding to the regulatory element of TSLP.5Harada M. Hirota T. Jodo A.I. Doi S. Kameda M. Fujita K. et al.Functional analysis of the thymic stromal lymphopoietin variants in human bronchial epithelial cells.Am J Respir Cell Mol Biol. 2009; 40: 368-374Crossref PubMed Scopus (127) Google Scholar SNP rs2416259 was also associated with risk of ADEH, as well as associated phenotypes, high tIgE concentrations, and EASI scores. Of interest, SNP rs2289276, an SNP previously associated with specific IgE levels to cockroach antigen and total IgE concentration,8Hunninghake G.M. Lasky-Su J. Soto-Quiros M.E. Avila L. Liang C. Lake S.L. et al.Sex-stratified linkage analysis identifies a female-specific locus for IgE to cockroach in Costa Ricans.Am J Respir Crit Care Med. 2008; 177: 830-836Crossref PubMed Scopus (62) Google Scholar was associated with AD in our study. Three validated coding dbSNPs in TSLPR were significantly associated with AD in European American subjects, one of which is a nonsense SNP (rs36177645 X201W), which might lead to premature termination of peptides; investigation into the effects on gene expression are underway. Potential epistatic effects between SNPs in TSLP and its 2 receptors, IL7R and TSLPR, were observed for both AD and ADEH. Associations for SNP-SNP interactions were stronger than the single-marker associations. A gene-for-gene replication of associations was observed in the African American sample for the traits analyzed (AD, tIgE concentration, and EASI score) and TSLP and IL7R SNPs. Because of the smaller African American ADEH sample, it was not possible to test for replication to the ADEH trait. However, the SNPs significantly associated with AD, tIgE concentration, and EASI score in the African American subjects were not for the same SNPs as those associated with these 3 traits in the European American subjects. Failure to observe SNP-for-SNP replication in ethnically diverse populations is not uncommon,9Cui Y. Kang

<h3>Background</h3> The genetic determinants of the human innate immune response are poorly understood. Apolipoprotein (Apo) E, a lipid-trafficking protein that affects inflammation, has well-described wild-type (ε3) and disease-associated (ε2 and ε4) alleles, but its connection to human innate immunity is undefined. <h3>Objective</h3> We sought to define the relationship of <i>APOε4</i> to the human innate immune response. <h3>Methods</h3> We evaluated <i>APOε4</i> in several functional models of the human innate immune response, including intravenous LPS challenge in human subjects, and assessed <i>APOε4</i> association to organ injury in patients with severe sepsis, a disease driven by dysregulated innate immunity. <h3>Results</h3> Whole blood from healthy <i>APOε3</i>/<i>APOε4</i> volunteers induced higher cytokine levels on <i>ex vivo</i> stimulation with Toll-like receptor (TLR) 2, TLR4, or TLR5 ligands than blood from <i>APOε3</i>/<i>APOε3</i> patients, whereas TLR7/8 responses were similar. This was associated with increased lipid rafts in <i>APOε3/APOε4</i> monocytes. By contrast, <i>APOε3</i>/<i>APOε3</i> and <i>APOε3</i>/<i>APOε4</i> serum neutralized LPS equivalently and supported similar LPS responses in <i>Apoe</i>-deficient macrophages, arguing against a differential role for secretory APOE4 protein. After intravenous LPS, <i>APOε3/APOε4</i> patients had higher hyperthermia and plasma TNF-α levels and earlier plasma IL-6 than <i>APOε3/APOε3</i> patients. APOE4-targeted replacement mice displayed enhanced hypothermia, plasma cytokines, and hepatic injury and altered splenic lymphocyte apoptosis after systemic LPS compared with APOE3 counterparts. In a cohort of 828 patients with severe sepsis, <i>APOε4</i> was associated with increased coagulation system failure among European American patients. <h3>Conclusions</h3> <i>APOε4</i> is a determinant of the human innate immune response to multiple TLR ligands and associates with altered patterns of organ injury in human sepsis.

Genome-wide association studies (GWAS) have emerged as powerful means for identifying genetic loci related to complex diseases. However, the role of environment and its potential to interact with key loci has not been adequately addressed in most GWAS. Networks of collaborative studies involving different study populations and multiple phenotypes provide a powerful approach for addressing the challenges in analysis and interpretation shared across studies. The Gene, Environment Association Studies (GENEVA) consortium was initiated to: identify genetic variants related to complex diseases; identify variations in gene-trait associations related to environmental exposures; and ensure rapid sharing of data through the database of Genotypes and Phenotypes. GENEVA consists of several academic institutions, including a coordinating center, two genotyping centers and 14 independently designed studies of various phenotypes, as well as several Institutes and Centers of the National Institutes of Health led by the National Human Genome Research Institute. Minimum detectable effect sizes include relative risks ranging from 1.24 to 1.57 and proportions of variance explained ranging from 0.0097 to 0.02. Given the large number of research participants (N>80,000), an important feature of GENEVA is harmonization of common variables, which allow analyses of additional traits. Environmental exposure information available from most studies also enables testing of gene-environment interactions. Facilitated by its sizeable infrastructure for promoting collaboration, GENEVA has established a unified framework for genotyping, data quality control, analysis and interpretation. By maximizing knowledge obtained through collaborative GWAS incorporating environmental exposure information, GENEVA aims to enhance our understanding of disease etiology, potentially identifying opportunities for intervention.

Atopic dermatitis (AD) is characterized by epidermal tight junction (TJ) defects and a propensity for Staphylococcus aureus skin infections. S. aureus is sensed by many pattern recognition receptors, including Toll-like receptor 2 (TLR2). We hypothesized that an effective innate immune response will include skin barrier repair, and that this response is impaired in AD subjects. S. aureus-derived peptidoglycan (PGN) and synthetic TLR2 agonists enhanced TJ barrier and increased expression of TJ proteins, claudin-1 (CLDN1), claudin-23 (CLDN23), occludin, and Zonulae occludens 1 (ZO-1) in primary human keratinocytes. A TLR2 agonist enhanced skin barrier recovery in human epidermis wounded by tape stripping. Tlr2(-/-) mice had a delayed and incomplete barrier recovery following tape stripping. AD subjects had reduced epidermal TLR2 expression as compared with nonatopic subjects, which inversely correlated (r=-0.654, P=0.0004) with transepidermal water loss (TEWL). These observations indicate that TLR2 activation enhances skin barrier in murine and human skin and is an important part of a wound repair response. Reduced epidermal TLR2 expression observed in AD patients may have a role in their incompetent skin barrier.

The genetic basis of acute lung injury (ALI) is poorly understood. The myosin light chain kinase (MYLK) gene encodes the nonmuscle myosin light chain kinase isoform, a multifunctional protein involved in the inflammatory response (apoptosis, vascular permeability, leukocyte diapedesis). To examine MYLK as a novel candidate gene in sepsis-associated ALI, we sequenced exons, exon-intron boundaries, and 2 kb of 5' UTR of the MYLK, which revealed 51 single-nucleotide polymorphisms (SNPs). Potential association of 28 MYLK SNPs with sepsis-associated ALI were evaluated in a case-control sample of 288 European American subjects (EAs) with sepsis alone, subjects with sepsis-associated ALI, or healthy control subjects, and a sample population of 158 African American subjects (AAs) with sepsis and ALI. Significant single locus associations in EAs were observed between four MYLK SNPs and the sepsis phenotype (P<0.001), with an additional SNP associated with the ALI phenotype (P=0.03). A significant association of a single SNP (identical to the SNP identified in EAs) was observed in AAs with sepsis (P=0.002) and with ALI (P=0.01). Three sepsis risk-conferring haplotypes in EAs were defined downstream of start codon of smooth muscle MYLK isoform, a region containing putative regulatory elements (P<0.001). In contrast, multiple haplotypic analyses revealed an ALI-specific, risk-conferring haplotype at 5' of the MYLK gene in both European and African Americans and an additional 3' region haplotype only in African Americans. These data strongly implicate MYLK genetic variants to confer increased risk of sepsis and sepsis-associated ALI.

Abstract The African Diaspora in the Western Hemisphere represents one of the largest forced migrations in history and had a profound impact on genetic diversity in modern populations. To date, the fine-scale population structure of descendants of the African Diaspora remains largely uncharacterized. Here we present genetic variation from deeply sequenced genomes of 642 individuals from North and South American, Caribbean and West African populations, substantially increasing the lexicon of human genomic variation and suggesting much variation remains to be discovered in African-admixed populations in the Americas. We summarize genetic variation in these populations, quantifying the postcolonial sex-biased European gene flow across multiple regions. Moreover, we refine estimates on the burden of deleterious variants carried across populations and how this varies with African ancestry. Our data are an important resource for empowering disease mapping studies in African-admixed individuals and will facilitate gene discovery for diseases disproportionately affecting individuals of African ancestry.

Abstract Objective Protein citrullination is an important posttranslational modification recognized by rheumatoid arthritis (RA)–specific autoantibodies. One of the citrullinating enzymes, peptidyl arginine deiminase type 4 (PAD‐4), is genetically associated with development of RA in some populations, although the mechanism(s) mediating this effect are not yet clear. There have been descriptions of anti–PAD‐4 autoantibodies in different rheumatic diseases. This study was undertaken to investigate whether anti–PAD‐4 antibodies are specific to RA, are associated with disease phenotype or severity, and whether PAD‐4 polymorphisms influence the anti–PAD‐4 autoantibody response. Methods Sera from patients with established RA, patients with other rheumatic diseases, and healthy adults were assayed for anti–PAD‐4 autoantibodies by immunoprecipitation of in vitro–translated PAD‐4. The epitope(s) recognized by PAD‐4 autoantibodies were mapped using various PAD‐4 truncations. PAD‐4 genotyping was performed on RA patients with the TaqMan assay. Joint erosions were scored from hand and foot radiographs using the Sharp/van der Heijde method. Results PAD‐4 autoantibodies were found in 36–42% of RA patients, and were very infrequent in controls. Recognition by anti–PAD‐4 autoantibodies required the 119 N‐terminal amino acids, which encompass the 3 nonsynonymous polymorphisms associated with disease susceptibility. Strikingly, the anti–PAD‐4 immune response was associated with the RA susceptibility haplotype of PADI4 . Anti–PAD‐4 antibodies were associated with more severe joint destruction in RA. Conclusion Our findings indicate that anti–PAD‐4 antibodies are specific markers of RA, independently associated with more severe disease, suggesting that an anti–PAD‐4 immune response may be involved in pathways of joint damage in this disease. Polymorphisms in the PADI4 gene influence the immune response to the PAD‐4 protein, potentially contributing to disease propagation.

BackgroundThe basis for increased susceptibility of patients with atopic dermatitis (AD) to develop disseminated viral skin infections such as eczema herpeticum (AD with a history of eczema herpeticum, ADEH+) is poorly understood.ObjectiveWe sought to determine whether subjects with AD prone to disseminated viral skin infections have defects in their IFN responses.MethodsGeneChip profiling was used to identify differences in gene expression of PBMCs from patients with ADEH+ compared with patients with AD without a history of eczema herpeticum (ADEH–) and nonatopic controls. Key differences in protein expression were verified by enzyme-linked immunosorbent spot assay and/or ELISA. Clinical relevance was further demonstrated by a mouse model of disseminated viral skin infection and genetic association analysis for genetic variants in IFNG and IFNGR1 and ADEH among 435 cases and controls.ResultsWe demonstrate by global gene expression analysis selective transcriptomic changes within the IFN superfamily of PBMCs from subjects with ADEH+ reflecting low IFN-γ and IFN-γ receptor gene expression. IFN-γ protein production was also significantly lower in patients with ADEH+ (n = 24) compared with patients with ADEH– (n = 20) and nonatopic controls (n = 20). IFN-γ receptor knockout mice developed disseminated viral skin infection after epicutaneous challenge with vaccinia virus. Genetic variants in IFNG and IFNGR1 single nucleotide polymorphisms (SNPs) were significantly associated with ADEH (112 cases, 166 controls) and IFN-γ production: a 2-SNP (A-G) IFNGR1 haplotype (rs10457655 and rs7749390) showed the strongest association with a reduced risk of ADEH+ (13.2% ADEH+ vs 25.5% ADEH–; P = .00057).ConclusionPatients with ADEH+ have reduced IFN-γ production, and IFNG and IFNGR1 SNPs are significantly associated with ADEH+ and may contribute to an impaired immune response to herpes simplex virus. The basis for increased susceptibility of patients with atopic dermatitis (AD) to develop disseminated viral skin infections such as eczema herpeticum (AD with a history of eczema herpeticum, ADEH+) is poorly understood. We sought to determine whether subjects with AD prone to disseminated viral skin infections have defects in their IFN responses. GeneChip profiling was used to identify differences in gene expression of PBMCs from patients with ADEH+ compared with patients with AD without a history of eczema herpeticum (ADEH–) and nonatopic controls. Key differences in protein expression were verified by enzyme-linked immunosorbent spot assay and/or ELISA. Clinical relevance was further demonstrated by a mouse model of disseminated viral skin infection and genetic association analysis for genetic variants in IFNG and IFNGR1 and ADEH among 435 cases and controls. We demonstrate by global gene expression analysis selective transcriptomic changes within the IFN superfamily of PBMCs from subjects with ADEH+ reflecting low IFN-γ and IFN-γ receptor gene expression. IFN-γ protein production was also significantly lower in patients with ADEH+ (n = 24) compared with patients with ADEH– (n = 20) and nonatopic controls (n = 20). IFN-γ receptor knockout mice developed disseminated viral skin infection after epicutaneous challenge with vaccinia virus. Genetic variants in IFNG and IFNGR1 single nucleotide polymorphisms (SNPs) were significantly associated with ADEH (112 cases, 166 controls) and IFN-γ production: a 2-SNP (A-G) IFNGR1 haplotype (rs10457655 and rs7749390) showed the strongest association with a reduced risk of ADEH+ (13.2% ADEH+ vs 25.5% ADEH–; P = .00057). Patients with ADEH+ have reduced IFN-γ production, and IFNG and IFNGR1 SNPs are significantly associated with ADEH+ and may contribute to an impaired immune response to herpes simplex virus.

Arachidonic acid (AA) is a long-chain omega-6 polyunsaturated fatty acid (PUFA) synthesized from the precursor dihomo-gamma-linolenic acid (DGLA) that plays a vital role in immunity and inflammation. Variants in the Fatty Acid Desaturase (FADS) family of genes on chromosome 11q have been shown to play a role in PUFA metabolism in populations of European and Asian ancestry; no work has been done in populations of African ancestry to date.In this study, we report that African Americans have significantly higher circulating levels of plasma AA (p = 1.35 × 10(-48)) and lower DGLA levels (p = 9.80 × 10(-11)) than European Americans. Tests for association in N = 329 individuals across 80 nucleotide polymorphisms (SNPs) in the Fatty Acid Desaturase (FADS) locus revealed significant association with AA, DGLA and the AA/DGLA ratio, a measure of enzymatic efficiency, in both racial groups (peak signal p = 2.85 × 10(-16) in African Americans, 2.68 × 10(-23) in European Americans). Ancestry-related differences were observed at an upstream marker previously associated with AA levels (rs174537), wherein, 79-82% of African Americans carry two copies of the G allele compared to only 42-45% of European Americans. Importantly, the allelic effect of the G allele, which is associated with enhanced conversion of DGLA to AA, on enzymatic efficiency was similar in both groups.We conclude that the impact of FADS genetic variants on PUFA metabolism, specifically AA levels, is likely more pronounced in African Americans due to the larger proportion of individuals carrying the genotype associated with increased FADS1 enzymatic conversion of DGLA to AA.

Studies of recombination and how it varies depend crucially on accurate recombination maps. We propose a new approach for constructing high-resolution maps of relative recombination rates based on the observation of ancestry switch points among admixed individuals. We show the utility of this approach using simulations and by applying it to SNP genotype data from a sample of 2,565 African Americans and 299 African Caribbeans and detecting several hundred thousand recombination events. Comparison of the inferred map with high-resolution maps from non-admixed populations provides evidence of fine-scale differentiation in recombination rates between populations. Overall, the admixed map is well predicted by the average proportion of admixture and the recombination rate estimates from the source populations. The exceptions to this are in areas surrounding known large chromosomal structural variants, specifically inversions. These results suggest that outside of structurally variable regions, admixture does not substantially disrupt the factors controlling recombination rates in humans.

Single nucleotide polymorphisms (SNPs) in thymic stromal lymphopoietin (TSLP) have been associated with IgE (in girls) and asthma (in general). We sought to determine whether TSLP SNPs are associated with asthma in a sex-specific fashion.We conducted regular and sex-stratified analyses of association between SNPs in TSLP and asthma in families of children with asthma in Costa Rica. Significant findings were replicated in whites and African-American participants in the Childhood Asthma Management Program, in African-Americans in the Genomic Research on Asthma in the African Diaspora study, in whites and Hispanics in the Children's Health Study, and in whites in the Framingham Heart Study (FHS).Two SNPs in TSLP (rs1837253 and rs2289276) were significantly associated with a reduced risk of asthma in combined analyses of all cohorts (P values of 2 × 10(-5) and 1 × 10(-5) , respectively). In a sex-stratified analysis, the T allele of rs1837253 was significantly associated with a reduced risk of asthma in males only (P = 3 × 10(-6) ). Alternately, the T allele of rs2289276 was significantly associated with a reduced risk of asthma in females only (P = 2 × 10(-4) ). Findings for rs2289276 were consistent in all cohorts except the FHS.TSLP variants are associated with asthma in a sex-specific fashion.

Smoking while pregnant is associated with a myriad of negative health outcomes in the child. Some of the detrimental effects may be due to epigenetic modifications, although few studies have investigated this hypothesis in detail.To characterize site-specific epigenetic modifications conferred by prenatal smoking exposure within asthmatic children.Using Illumina HumanMethylation27 microarrays, we estimated the degree of methylation at 27,578 distinct DNA sequences located primarily in gene promoters using whole blood DNA samples from the Childhood Asthma Management Program (CAMP) subset of Asthma BRIDGE childhood asthmatics (n = 527) ages 5-12 with prenatal smoking exposure data available. Using beta-regression, we screened loci for differential methylation related to prenatal smoke exposure, adjusting for gender, age and clinical site, and accounting for multiple comparisons by FDR.Of 27,578 loci evaluated, 22,131 (80%) passed quality control assessment and were analyzed. Sixty-five children (12%) had a history of prenatal smoke exposure. At an FDR of 0.05, we identified 19 CpG loci significantly associated with prenatal smoke, of which two replicated in two independent populations. Exposure was associated with a 2% increase in mean CpG methylation in FRMD4A (p = 0.01) and Cllorf52 (p = 0.001) compared to no exposure. Four additional genes, XPNPEP1, PPEF2, SMPD3 and CRYGN, were nominally associated in at least one replication group.These data suggest that prenatal exposure to tobacco smoke is associated with reproducible epigenetic changes that persist well into childhood. However, the biological significance of these altered loci remains unknown.

Asthma is a growing public health problem in developing countries. However, few studies have studied the role of urbanisation in this phenomenon. It was hypothesised that children living in a peri-urban setting in Peru have higher rates of asthma and allergy than rural counterparts.1441 adolescents aged 13-15 years were enrolled from two settings: a peri-urban shanty town in Lima (n = 725) and 23 rural villages in Tumbes (n = 716). Participants filled in questionnaires on asthma and allergy symptoms, environmental exposures and sociodemographics, and underwent spirometry, and exhaled nitric oxide (eNO) and allergy skin testing. Indoor particulate matter (PM) concentrations were measured in 170 households.Lima adolescents had higher rates of lifetime wheezing (22% vs 10%), current asthma symptoms (12% vs 3%) and physician-diagnosed asthma (13% vs 2%; all p <0.001). Current rhinitis (23% vs 12%), eczema (12% vs 0.4%), atopy (56% vs 38%), personal history of cigarette smoking (7.4% vs 1.3%) and mean indoor PM (31 vs 13 μg/m(3)) were also higher in Lima (all p < 0.001). The peri-urban environment of Lima was associated with a 2.6-fold greater odds (95% CI 1.3 to 5.3) of asthma in multivariable regression. Forced expiratory volumes were higher and FEV(1)/FVC (forced expiratory volume in 1 s/forced vital capacity) ratios were lower in Lima (all p < 0.001). Higher eNO values in Lima (p < 0.001) were attributable to higher rates of asthma and atopy.Peri-urban adolescents had more asthma, atopy and airways inflammation and were exposed to more indoor pollution. The findings provide evidence of the risks posed to lung health by peri-urban environments in developing countries.

Stress is associated with asthma morbidity in Puerto Ricans (PRs), who have reduced bronchodilator response (BDR).To examine whether stress and/or a gene regulating anxiety (ADCYAP1R1) is associated with BDR in PR and non-PR children with asthma.This was a cross-sectional study of stress and BDR (percent change in FEV1 after BD) in 234 PRs ages 9-14 years with asthma. We assessed child stress using the Checklist of Children's Distress Symptoms, and maternal stress using the Perceived Stress Scale. Replication analyses were conducted in two cohorts. Polymorphisms in ADCYAP1R1 were genotyped in our study and six replication studies. Multivariable models of stress and BDR were adjusted for age, sex, income, environmental tobacco smoke, and use of inhaled corticosteroids.High child stress was associated with reduced BDR in three cohorts. PR children who were highly stressed (upper quartile, Checklist of Children's Distress Symptoms) and whose mothers had high stress (upper quartile, Perceived Stress Scale) had a BDR that was 10.2% (95% confidence interval, 6.1-14.2%) lower than children who had neither high stress nor a highly stressed mother. A polymorphism in ADCYAP1R1 (rs34548976) was associated with reduced BDR. This single-nucleotide polymorphism is associated with reduced expression of the gene for the β2-adrenergic receptor (ADRB2) in CD4(+) lymphocytes of subjects with asthma, and it affects brain connectivity of the amygdala and the insula (a biomarker of anxiety).High child stress and an ADCYAP1R1 single-nucleotide polymorphism are associated with reduced BDR in children with asthma. This is likely caused by down-regulation of ADRB2 in highly stressed children.

Long chain polyunsaturated fatty acids (LC-PUFAs) are essential for brain structure, development, and function, and adequate dietary quantities of LC-PUFAs are thought to have been necessary for both brain expansion and the increase in brain complexity observed during modern human evolution. Previous studies conducted in largely European populations suggest that humans have limited capacity to synthesize brain LC-PUFAs such as docosahexaenoic acid (DHA) from plant-based medium chain (MC) PUFAs due to limited desaturase activity. Population-based differences in LC-PUFA levels and their product-to-substrate ratios can, in part, be explained by polymorphisms in the fatty acid desaturase (FADS) gene cluster, which have been associated with increased conversion of MC-PUFAs to LC-PUFAs. Here, we show evidence that these high efficiency converter alleles in the FADS gene cluster were likely driven to near fixation in African populations by positive selection ∼85 kya. We hypothesize that selection at FADS variants, which increase LC-PUFA synthesis from plant-based MC-PUFAs, played an important role in allowing African populations obligatorily tethered to marine sources for LC-PUFAs in isolated geographic regions, to rapidly expand throughout the African continent 60–80 kya.

Asthma is a common chronic disease without cure. Our understanding of asthma onset, pathobiology, classification, and management has evolved substantially over the past decade; however, significant asthma-related morbidity and excess healthcare use and costs persist. To address this important clinical condition, the NHLBI convened a group of extramural investigators for an Asthma Research Strategic Planning workshop on September 18-19, 2014, to accelerate discoveries and their translation to patients. The workshop focused on (1) in utero and early-life origins of asthma, (2) the use of phenotypes and endotypes to classify disease, (3) defining disease modification, (4) disease management, and (5) implementation research. This report summarizes the workshop and produces recommendations to guide future research in asthma.

Pulmonary arterial hypertension (PAH) is a medically incurable disease resulting in death from right ventricular (RV) failure. Both pulmonary vascular and RV remodeling are linked to dynamic changes in the microvasculature. Therefore, we hypothesized that circulating angiostatic factors could be linked to outcomes and represent novel biomarkers of disease severity in PAH.We sought to determine the relationship of a potent angiostatic factor, endostatin (ES), with disease severity and mortality in PAH. Furthermore, we assessed genetic predictors of ES expression and/or function and their association with outcomes in PAH.We measured levels of serum ES in two independent cohorts of patients with PAH. Contemporaneous clinical data included New York Heart Association functional class, 6-minute-walk distance, invasive hemodynamics, and laboratory chemistries.Serum ES correlated with poor functional status, decreased exercise tolerance, and invasive hemodynamics variables. Furthermore, serum ES was a strong predictor of mortality. A loss-of-function, missense variant in the gene encoding ES, Col18a1, was linked to lower circulating protein and was independently associated with reduced mortality.Our data link increased expression of ES to disease severity in PAH and demonstrate a significant relationship with adverse outcomes. Circulating ES levels can be genetically influenced, implicating ES as a genetically determined modifier of disease severity impacting on survival. These observations support serum ES as a potential biomarker in PAH with the capacity to predict poor outcomes. More importantly, this study implicates Col18a1/ES as a potential new therapeutic target in PAH.

The airway transcriptome includes genes that contribute to the pathophysiologic heterogeneity seen in individuals with asthma.We analyzed sputum gene expression for transcriptomic endotypes of asthma (TEA), gene signatures that discriminate phenotypes of disease.Gene expression in the sputum and blood of patients with asthma was measured using Affymetrix microarrays. Unsupervised clustering analysis based on pathways from the Kyoto Encyclopedia of Genes and Genomes was used to identify TEA clusters. Logistic regression analysis of matched blood samples defined an expression profile in the circulation to determine the TEA cluster assignment in a cohort of children with asthma to replicate clinical phenotypes.Three TEA clusters were identified. TEA cluster 1 had the most subjects with a history of intubation (P = 0.05), a lower prebronchodilator FEV1 (P = 0.006), a higher bronchodilator response (P = 0.03), and higher exhaled nitric oxide levels (P = 0.04) compared with the other TEA clusters. TEA cluster 2, the smallest cluster, had the most subjects that were hospitalized for asthma (P = 0.04). TEA cluster 3, the largest cluster, had normal lung function, low exhaled nitric oxide levels, and lower inhaled steroid requirements. Evaluation of TEA clusters in children confirmed that TEA clusters 1 and 2 are associated with a history of intubation (P = 5.58 × 10(-6)) and hospitalization (P = 0.01), respectively.There are common patterns of gene expression in the sputum and blood of children and adults that are associated with near-fatal, severe, and milder asthma.

Abstract Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA , and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans.

De novo mutations (DNMs), or mutations that appear in an individual despite not being seen in their parents, are an important source of genetic variation whose impact is relevant to studies of human evolution, genetics, and disease. Utilizing high-coverage whole-genome sequencing data as part of the Trans-Omics for Precision Medicine (TOPMed) Program, we called 93,325 single-nucleotide DNMs across 1,465 trios from an array of diverse human populations, and used them to directly estimate and analyze DNM counts, rates, and spectra. We find a significant positive correlation between local recombination rate and local DNM rate, and that DNM rate explains a substantial portion (8.98 to 34.92%, depending on the model) of the genome-wide variation in population-level genetic variation from 41K unrelated TOPMed samples. Genome-wide heterozygosity does correlate with DNM rate, but only explains &lt;1% of variation. While we are underpowered to see small differences, we do not find significant differences in DNM rate between individuals of European, African, and Latino ancestry, nor across ancestrally distinct segments within admixed individuals. However, we did find significantly fewer DNMs in Amish individuals, even when compared with other Europeans, and even after accounting for parental age and sequencing center. Specifically, we found significant reductions in the number of C→A and T→C mutations in the Amish, which seem to underpin their overall reduction in DNMs. Finally, we calculated near-zero estimates of narrow sense heritability ( h 2 ), which suggest that variation in DNM rate is significantly shaped by nonadditive genetic effects and the environment.

To characterize the extent and impact of ancestry-related biases in precision genomic medicine, we use 642 whole-genome sequences from the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) project to evaluate typical filters and databases. We find significant correlations between estimated African ancestry proportions and the number of variants per individual in all variant classification sets but one. The source of these correlations is highlighted in more detail by looking at the interaction between filtering criteria and the ClinVar and Human Gene Mutation databases. ClinVar's correlation, representing African ancestry-related bias, has changed over time amidst monthly updates, with the most extreme switch happening between March and April of 2014 (r=0.733 to r=-0.683). We identify 68 SNPs as the major drivers of this change in correlation. As long as ancestry-related bias when using these clinical databases is minimally recognized, the genetics community will face challenges with implementation, interpretation and cost-effectiveness when treating minority populations.

To the Editor: Risk variants in the APOL1 gene on chromosome 22, which were first discovered in African Americans, confer a substantially increased risk of kidney disease, early-onset hypertension, and cardiovascular disease, although disease risk is modified by other genetic factors and by environmental factors. 1 This is an important discovery for genomic medicine and has improved our understanding of disparities among ethnic groups. 2 Efforts are under way to incorporate APOL1 testing in routine clinical settings, and APOL1 is under consideration as a therapeutic target for kidney disease. 3he APOL1 risk variants rose to high frequency in Western Africa in response to trypanosomal infection, 4 and most studies of APOL1 have involved populations of persons who self-report as African or African American.However, other populations who also share recent ancestry from Africa, such as Hispanic populations, may be at greater risk than expected for APOL1-driven disease.These persons may not undergo testing; however, they may still be at high risk because of the presence of APOL1 risk variants. 5Thus, it is important to understand more fully the global distribution of these variants on the basis of country of origin and genetic ancestry rather than self-reported race or ethnic group.We used linked genetic and demographic data from 111 populations in two large studies, the Population Architecture using Genomics and Epidemiology Study (https://pagestudy .org) and the Consortium on Asthma among African-ancestry Populations in the Americas (www .caapa -project .org), to determine the global frequencies of APOL1 risk variants (see the Supplementary Appendix, available with the full text of this letter at NEJM.org).We inferred risk-allele status using the two G1 alleles (rs60910145 and rs73885319) and the proxy single-nucleotide polymorphism commonly typed for G2 (rs12106505).The frequencies of the recessive risk genotypes

Asthma is a complex disease with striking disparities across racial and ethnic groups. Despite its relatively high burden, representation of individuals of African ancestry in asthma genome-wide association studies (GWAS) has been inadequate, and true associations in these underrepresented minority groups have been inconclusive. We report the results of a genome-wide meta-analysis from the Consortium on Asthma among African Ancestry Populations (CAAPA; 7009 asthma cases, 7645 controls). We find strong evidence for association at four previously reported asthma loci whose discovery was driven largely by non-African populations, including the chromosome 17q12-q21 locus and the chr12q13 region, a novel (and not previously replicated) asthma locus recently identified by the Trans-National Asthma Genetic Consortium (TAGC). An additional seven loci reported by TAGC show marginal evidence for association in CAAPA. We also identify two novel loci (8p23 and 8q24) that may be specific to asthma risk in African ancestry populations.

Genetic factors influence chronic obstructive pulmonary disease (COPD) risk, but the individual variants that have been identified have small effects. We hypothesised that a polygenic risk score using additional variants would predict COPD and associated phenotypes.We constructed a polygenic risk score using a genome-wide association study of lung function (FEV1 and FEV1/forced vital capacity [FVC]) from the UK Biobank and SpiroMeta. We tested this polygenic risk score in nine cohorts of multiple ethnicities for an association with moderate-to-severe COPD (defined as FEV1/FVC <0·7 and FEV1 <80% of predicted). Associations were tested using logistic regression models, adjusting for age, sex, height, smoking pack-years, and principal components of genetic ancestry. We assessed predictive performance of models by area under the curve. In a subset of studies, we also studied quantitative and qualitative CT imaging phenotypes that reflect parenchymal and airway pathology, and patterns of reduced lung growth.The polygenic risk score was associated with COPD in European (odds ratio [OR] per SD 1·81 [95% CI 1·74-1·88] and non-European (1·42 [1·34-1·51]) populations. Compared with the first decile, the tenth decile of the polygenic risk score was associated with COPD, with an OR of 7·99 (6·56-9·72) in European ancestry and 4·83 (3·45-6·77) in non-European ancestry cohorts. The polygenic risk score was superior to previously described genetic risk scores and, when combined with clinical risk factors (ie, age, sex, and smoking pack-years), showed improved prediction for COPD compared with a model comprising clinical risk factors alone (AUC 0·80 [0·79-0·81] vs 0·76 [0·75-0·76]). The polygenic risk score was associated with CT imaging phenotypes, including wall area percent, quantitative and qualitative measures of emphysema, local histogram emphysema patterns, and destructive emphysema subtypes. The polygenic risk score was associated with a reduced lung growth pattern.A risk score comprised of genetic variants can identify a small subset of individuals at markedly increased risk for moderate-to-severe COPD, emphysema subtypes associated with cigarette smoking, and patterns of reduced lung growth.US National Institutes of Health, Wellcome Trust.

Gauging the spectrum of human mutations It has become increasing clear that mutation affects phenotypic variation and disease risk across humans. However, there are many different types of mutation. Seplyarskiy et al . applied a matrix factorization method to large human genomic datasets to identify germline mutational processes in an unsupervised manner. From this survey, nine robust mutational components were identified and specific mechanisms generating seven of these processes are proposed from correlations with genomic features. These results confirm and improve upon our understanding of mutational processes and reveal likely mechanisms of mutation in the human genome. —LMZ

Abstract Genotype-phenotype association studies often combine phenotype data from multiple studies to increase statistical power. Harmonization of the data usually requires substantial effort due to heterogeneity in phenotype definitions, study design, data collection procedures, and data-set organization. Here we describe a centralized system for phenotype harmonization that includes input from phenotype domain and study experts, quality control, documentation, reproducible results, and data-sharing mechanisms. This system was developed for the National Heart, Lung, and Blood Institute’s Trans-Omics for Precision Medicine (TOPMed) program, which is generating genomic and other -omics data for more than 80 studies with extensive phenotype data. To date, 63 phenotypes have been harmonized across thousands of participants (recruited in 1948–2012) from up to 17 studies per phenotype. Here we discuss challenges in this undertaking and how they were addressed. The harmonized phenotype data and associated documentation have been submitted to National Institutes of Health data repositories for controlled access by the scientific community. We also provide materials to facilitate future harmonization efforts by the community, which include 1) the software code used to generate the 63 harmonized phenotypes, enabling others to reproduce, modify, or extend these harmonizations to additional studies, and 2) the results of labeling thousands of phenotype variables with controlled vocabulary terms.

Human genetic studies support an inverse causal relationship between leukocyte telomere length (LTL) and coronary artery disease (CAD), but directionally mixed effects for LTL and diverse malignancies. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by expansion of hematopoietic cells bearing leukemogenic mutations, predisposes both hematologic malignancy and CAD. TERT (which encodes telomerase reverse transcriptase) is the most significantly associated germline locus for CHIP in genome-wide association studies. Here, we investigated the relationship between CHIP, LTL, and CAD in the Trans-Omics for Precision Medicine (TOPMed) program (n = 63,302) and UK Biobank (n = 47,080). Bidirectional Mendelian randomization studies were consistent with longer genetically imputed LTL increasing propensity to develop CHIP, but CHIP then, in turn, hastens to shorten measured LTL (mLTL). We also demonstrated evidence of modest mediation between CHIP and CAD by mLTL. Our data promote an understanding of potential causal relationships across CHIP and LTL toward prevention of CAD.

Summary Biobanks are being established across the world to understand the genetic, environmental, and epidemiological basis of human diseases with the goal of better prevention and treatments. Genome-wide association studies (GWAS) have been very successful at mapping genomic loci for a wide range of human diseases and traits, but in general, lack appropriate representation of diverse ancestries - with most biobanks and preceding GWAS studies composed of individuals of European ancestries. Here, we introduce the Global Biobank Meta-analysis Initiative (GBMI) -- a collaborative network of 19 biobanks from 4 continents representing more than 2.1 million consented individuals with genetic data linked to electronic health records. GBMI meta-analyzes summary statistics from GWAS generated using harmonized genotypes and phenotypes from member biobanks. GBMI brings together results from GWAS analysis across 6 main ancestry groups: approximately 33,000 of African ancestry either from Africa or from admixed-ancestry diaspora (AFR), 18,000 admixed American (AMR), 31,000 Central and South Asian (CSA), 341,000 East Asian (EAS), 1.4 million European (EUR), and 1,600 Middle Eastern (MID) individuals. In this flagship project, we generated GWASs from across 14 exemplar diseases and endpoints, including both common and less prevalent diseases that were previously understudied. Using the genetic association results, we validate that GWASs conducted in biobanks worldwide can be successfully integrated despite heterogeneity in case definitions, recruitment strategies, and baseline characteristics between biobanks. We demonstrate the value of this collaborative effort to improve GWAS power for diseases, increase representation, benefit understudied diseases, and improve risk prediction while also enabling the nomination of disease genes and drug candidates by incorporating gene and protein expression data and providing insight into the underlying biology of the studied traits.

Background: Plasma proteins are critical mediators of cardiovascular processes and are the targets of many drugs. Previous efforts to characterize the genetic architecture of the plasma proteome have been limited by a focus on individuals of European descent and leveraged genotyping arrays and imputation. Here we describe whole genome sequence analysis of the plasma proteome in individuals with greater African ancestry, increasing our power to identify novel genetic determinants. Methods: Proteomic profiling of 1301 proteins was performed in 1852 Black adults from the Jackson Heart Study using aptamer-based proteomics (SomaScan). Whole genome sequencing association analysis was ascertained for all variants with minor allele count ≥5. Results were validated using an alternative, antibody-based, proteomic platform (Olink) as well as replicated in the Multi-Ethnic Study of Atherosclerosis and the HERITAGE Family Study (Health, Risk Factors, Exercise Training and Genetics). Results: We identify 569 genetic associations between 479 proteins and 438 unique genetic regions at a Bonferroni-adjusted significance level of 3.8×10 -11 . These associations include 114 novel locus-protein relationships and an additional 217 novel sentinel variant-protein relationships. Novel cardiovascular findings include new protein associations at the APOE gene locus including ZAP70 (sentinel single nucleotide polymorphism [SNP] rs7412-T, β=0.61±0.05, P =3.27×10 -30 ) and MMP-3 (β=-0.60±0.05, P =1.67×10 -32 ), as well as a completely novel pleiotropic locus at the HPX gene, associated with 9 proteins. Further, the associations suggest new mechanisms of genetically mediated cardiovascular disease linked to African ancestry; we identify a novel association between variants linked to APOL1-associated chronic kidney and heart disease and the protein CKAP2 (rs73885319-G, β=0.34±0.04, P =1.34×10 -17 ) as well as an association between ATTR amyloidosis and RBP4 levels in community-dwelling individuals without heart failure. Conclusions: Taken together, these results provide evidence for the functional importance of variants in non-European populations, and suggest new biological mechanisms for ancestry-specific determinants of lipids, coagulation, and myocardial function.

Since the onset of the SARS-CoV-2 pandemic, most clinical testing has focused on RT-PCR1. Host epigenome manipulation post coronavirus infection2-4 suggests that DNA methylation signatures may differentiate patients with SARS-CoV-2 infection from uninfected individuals, and help predict COVID-19 disease severity, even at initial presentation.We customized Illumina's Infinium MethylationEPIC array to enhance immune response detection and profiled peripheral blood samples from 164 COVID-19 patients with longitudinal measurements of disease severity and 296 patient controls.Epigenome-wide association analysis revealed 13,033 genome-wide significant methylation sites for case-vs-control status. Genes and pathways involved in interferon signaling and viral response were significantly enriched among differentially methylated sites. We observe highly significant associations at genes previously reported in genetic association studies (e.g. IRF7, OAS1). Using machine learning techniques, models built using sparse regression yielded highly predictive findings: cross-validated best fit AUC was 93.6% for case-vs-control status, and 79.1%, 80.8%, and 84.4% for hospitalization, ICU admission, and progression to death, respectively.In summary, the strong COVID-19-specific epigenetic signature in peripheral blood driven by key immune-related pathways related to infection status, disease severity, and clinical deterioration provides insights useful for diagnosis and prognosis of patients with viral infections.Viral infections affect the body in many ways, including via changes to the epigenome, the sum of chemical modifications to an individual’s collection of genes that affect gene activity. Here, we analyzed the epigenome in blood samples from people with and without COVID-19 to determine whether we could find changes consistent with SARS-CoV-2 infection. Using a combination of statistical and machine learning techniques, we identify markers of SARS-CoV-2 infection as well as of severity and progression of COVID-19 disease. These signals of disease progression were present from the initial blood draw when first walking into the hospital. Together, these approaches demonstrate the potential of measuring the epigenome for monitoring SARS-CoV-2 status and severity.

Large-scale whole-genome sequencing studies have enabled analysis of noncoding rare-variant (RV) associations with complex human diseases and traits. Variant-set analysis is a powerful approach to study RV association. However, existing methods have limited ability in analyzing the noncoding genome. We propose a computationally efficient and robust noncoding RV association detection framework, STAARpipeline, to automatically annotate a whole-genome sequencing study and perform flexible noncoding RV association analysis, including gene-centric analysis and fixed window-based and dynamic window-based non-gene-centric analysis by incorporating variant functional annotations. In gene-centric analysis, STAARpipeline uses STAAR to group noncoding variants based on functional categories of genes and incorporate multiple functional annotations. In non-gene-centric analysis, STAARpipeline uses SCANG-STAAR to incorporate dynamic window sizes and multiple functional annotations. We apply STAARpipeline to identify noncoding RV sets associated with four lipid traits in 21,015 discovery samples from the Trans-Omics for Precision Medicine (TOPMed) program and replicate several of them in an additional 9,123 TOPMed samples. We also analyze five non-lipid TOPMed traits.

Meta-analysis is pervasively used to combine multiple genome-wide association studies (GWASs). Fine-mapping of meta-analysis studies is typically performed as in a single-cohort study. Here, we first demonstrate that heterogeneity (e.g., of sample size, phenotyping, imputation) hurts calibration of meta-analysis fine-mapping. We propose a summary statistics-based quality-control (QC) method, suspicious loci analysis of meta-analysis summary statistics (SLALOM), that identifies suspicious loci for meta-analysis fine-mapping by detecting outliers in association statistics. We validate SLALOM in simulations and the GWAS Catalog. Applying SLALOM to 14 meta-analyses from the Global Biobank Meta-analysis Initiative (GBMI), we find that 67% of loci show suspicious patterns that call into question fine-mapping accuracy. These predicted suspicious loci are significantly depleted for having nonsynonymous variants as lead variant (2.7×; Fisher's exact p = 7.3 × 10-4). We find limited evidence of fine-mapping improvement in the GBMI meta-analyses compared with individual biobanks. We urge extreme caution when interpreting fine-mapping results from meta-analysis of heterogeneous cohorts.

Precision medicine initiatives across the globe have led to a revolution of repositories linking large-scale genomic data with electronic health records, enabling genomic analyses across the entire phenome. Many of these initiatives focus solely on research insights, leading to limited direct benefit to patients. We describe the biobank at the Colorado Center for Personalized Medicine (CCPM Biobank) that was jointly developed by the University of Colorado Anschutz Medical Campus and UCHealth to serve as a unique, dual-purpose research and clinical resource accelerating personalized medicine. This living resource currently has more than 200,000 participants with ongoing recruitment. We highlight the clinical, laboratory, regulatory, and HIPAA-compliant informatics infrastructure along with our stakeholder engagement, consent, recontact, and participant engagement strategies. We characterize aspects of genetic and geographic diversity unique to the Rocky Mountain region, the primary catchment area for CCPM Biobank participants. We leverage linked health and demographic information of the CCPM Biobank participant population to demonstrate the utility of the CCPM Biobank to replicate complex trait associations in the first 33,674 genotyped individuals across multiple disease domains. Finally, we describe our current efforts toward return of clinical genetic test results, including high-impact pathogenic variants and pharmacogenetic information, and our broader goals as the CCPM Biobank continues to grow. Bringing clinical and research interests together fosters unique clinical and translational questions that can be addressed from the large EHR-linked CCPM Biobank resource within a HIPAA- and CLIA-certified environment.

Both a functional promoter polymorphism in the gene encoding CD14 (C-260T) and exposure to endotoxin are believed to play key roles in modulating the immune response and expression of atopic disease.We aimed to evaluate the role of the CD14 C-260T polymorphism in a population of African descent and to test for interaction between this genotype and house dust endotoxin (HDE) exposure on atopic phenotypes.Asthmatic probands and their families were recruited as part of the Barbados Asthma Genetics Study. The C-260T polymorphism and two additional CD14 promoter markers (G-1461T, C-1721T) were genotyped. Endotoxin was measured in house dust samples.Using a Family-Based Association Test, the C-260T allele appeared to be protective against asthma ( z = -2.444; P = .015) and asthma severity ( z = -2.615; P = .009) under a recessive model. No significant associations were observed for the G-1461T and C-1721T markers both individually and in haplotypes. In a case-control analysis, the CD14 TT genotype was found to reduce risk of asthma compared with the CD14 CC/CT genotypes (odds ratio [OR], 0.26; 95% CI, 0.14-0.49) and was associated with lower asthma severity scores ( P < .002). The TT genotype might protect against asthma for individuals with low HDE (OR, 0.09; 95% CI, 0.03-0.24), but may be a risk factor for individuals with high HDE (OR, 11.66; 95% CI, 1.03-131.7), suggesting a gene-environment interaction.These data suggest that the CD14-260 polymorphism may play a role in controlling risk to atopic disease and underscore the importance of incorporating key environmental exposures into studies of genetic risk factors.

Our objective was to characterize the pharmacokinetic properties of sulfadoxine-pyrimethamine in African adults and children with acute falciparum malaria. Despite decades of widespread use, there are few data to inform dose recommendations.In a prospective multicenter pharmacokinetic study in 307 patients with acute falciparum malaria, capillary blood concentrations of sulfadoxine and pyrimethamine were determined at 9 visits over a period of 42 days by mass spectrometry.After adjustment for dose, the area under the concentration-time curves (AUCs) of sulfadoxine and pyrimethamine in children aged 2 to 5 years were half of those in adults (median AUC, 410 microg/mL x d [interquartile range (IQR), 126-705 microg/mL x d] versus 816 microg/mL x d [IQR, 536-1150 microg/mL x d] [P = .0001] for sulfadoxine and 620 ng/mL x d [IQR, 229-1399 ng/mL x d] versus 1518 ng/mL x d [IQR, 1117-2013 ng/mL x d] for pyrimethamine). The effect of age on the AUC of sulfadoxine and pyrimethamine reflected higher clearance rates and larger apparent volumes of distribution in children aged 2 to 5 years when compared with adults (median clearance, 64.5 mL x kg(-1) x d(-1) [IQR, 46.2-132.6 mL x kg(-1) x d(-1)] versus 32.7 mL x kg(-1) x d(-1) [IQR, 22.3-52.2 mL x kg(-1) x d(-1)] for sulfadoxine [P = .0001] and 1.77 L x kg(-1) x d(-1) [IQR, 1.0-3.0 L x kg(-1) x d(-1)] versus 0.85 L x kg(-1) x d(-1) [IQR, 0.62-1.21 L x kg(-1) x d(-1)] for pyrimethamine [P = .0001]; median volume of distribution, 413 mL/kg [IQR, 299-711 mL/kg] versus 372 mL/kg [IQR, 267-488 mL/kg] for sulfadoxine [P = .0021] and 6.28 L/kg [IQR, 3.83-11.24 L/kg] versus 3.83 L/kg [IQR, 2.73-5.11 L/kg] for pyrimethamine [P = .0001]). Day 7 concentrations of both sulfadoxine and pyrimethamine provided good surrogate measures (R(2) >or= 0.72) of their respective AUCs.Pharmacokinetic factors may contribute to the increased risk of sulfadoxine-pyrimethamine antimalarial treatment failure in young children. The current dose recommendations need revision. We predict that children aged 2 to 5 years should be treated with 1 g sulfadoxine/50 mg pyrimethamine to achieve drug concentrations equivalent to those in adults.

Mechanical ventilation (MV) is an indispensable therapy for critically ill patients with acute lung injury and the adult respiratory distress syndrome. However, the mechanisms by which conventional MV induces lung injury remain unclear.We hypothesized that disruption of the gene encoding Nrf2, a transcription factor that regulates the induction of several antioxidant enzymes, enhances susceptibility to ventilator-induced lung injury (VILI) and that antioxidant supplementation attenuates this effect.To test our hypothesis and to examine the relevance of oxidative stress in VILI, we assessed lung injury and inflammatory responses in Nrf2-deficient (Nrf2(-/-)) mice and wild-type (Nrf2(+/+)) mice after an acute (2-h) injurious model of MV with or without administration of antioxidant.Nrf2(-/-) mice displayed greater levels of lung alveolar and vascular permeability and inflammatory responses to MV as compared with Nrf2(+/+) mice. Nrf2 deficiency enhances the levels of several proinflammatory cytokines implicated in the pathogenesis of VILI. We found diminished levels of critical antioxidant enzymes and redox imbalance by MV in the lungs of Nrf2(-/-) mice; however, antioxidant supplementation to Nrf2(-/-) mice remarkably attenuated VILI. When subjected to a clinically relevant prolong period of MV, Nrf2(-/-) mice displayed greater levels of VILI than Nrf2(+/+) mice. Expression profiling revealed lack of induction of several VILI genes, stress response and solute carrier proteins, and phosphatases in Nrf2(-/-) mice.Our data demonstrate for the first time a critical role for Nrf2 in VILI, which confers protection against cellular responses induced by MV by modulating oxidative stress.

It has been well established that acute lung injury (ALI), and the more severe presentation of acute respiratory distress syndrome (ARDS), constitute complex traits characterized by a multigenic and multifactorial etiology. Identification and validation of genetic variants contributing to disease susceptibility and severity has been hampered by the profound heterogeneity of the clinical phenotype and the role of environmental factors, which includes treatment, on outcome. The critical nature of ALI and ARDS, compounded by the impact of phenotypic heterogeneity, has rendered the amassing of sufficiently powered studies especially challenging. Nevertheless, progress has been made in the identification of genetic variants in select candidate genes, which has enhanced our understanding of the specific pathways involved in disease manifestation. Identification of novel candidate genes for which genetic association studies have confirmed a role in disease has been greatly aided by the powerful tool of high-throughput expression profiling. This article will review these studies to date, summarizing candidate genes associated with ALI and ARDS, acknowledging those that have been replicated in independent populations, with a special focus on the specific pathways for which candidate genes identified so far can be clustered.

To the Editor: Atopic dermatitis (AD) is the most common inflammatory skin disease, affecting up to 20% of children in the United States, and is characterized by an increased susceptibility to cutaneous infections.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 2Boguniewicz M. Leung D.Y. Recent insights into atopic dermatitis and implications for management of infectious complications.J Allergy Clin Immunol. 2010; 125: 4-13Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar One in 10 subjects with AD has difficulty clearing cutaneous infections with a host of viruses including herpes simplex, vaccinia, human papilloma, and/or molluscum contagiosum.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar This typically manifests as more extensive cutaneous and sometimes systemic disease and/or resistance to standard therapies. One of the rarer but more severe viral complications is eczema herpeticum (EH), caused by herpes simplex virus (HSV)–1 that results in a widespread skin infection.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar EH can be complicated by viremia and multiorgan involvement including keratoconjunctivitis, meningitis, and encephalitis.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Although we have shown that subjects with AD with more severe disease, greater allergen sensitization, and enhanced TH2 polarity are at greatest risk for this viral complication, the molecular mechanisms responsible for this remain unclear.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Disruption of the epidermal barrier is now recognized as a cardinal feature of AD pathogenesis. The skin has 2 barrier structures: the stratum corneum (SC) and tight junctions (TJs). It is widely accepted that the SC in subjects with AD is dysfunctional. The loss-of-function mutations in filaggrin, a structural protein important for its hygroscopic properties, have been reproducibly and robustly associated with AD3O’Regan G.M. Sandilands A. McLean W.H. Irvine A.D. Filaggrin in atopic dermatitis.J Allergy Clin Immunol. 2009; 124: R2-R6Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar and more recently with EH.4Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 507-513Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar TJs are found on opposing membranes of stratum granulosum keratinocytes, directly below the SC. TJs consist of a complex of adhesive and scaffolding proteins that control the passage of water, ions, and solutes through the paracellular pathway.5Niessen C.M. Tight junctions/adherens junctions: basic structure and function.J Invest Dermatol. 2007; 127: 2525-2532Crossref PubMed Scopus (508) Google Scholar We have recently demonstrated that the nonlesional epithelium of subjects with AD has remarkable bioelectric abnormalities indicative of a TJ defect that may be the consequence of reduced levels of claudin-1 (CLDN1), a key TJ adhesive protein.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar This is in line with the landmark work of Furuse and Tsukita,7Furuse M. Hata M. Furuse K. Yoshida Y. Haratake A. Sugitani Y. et al.Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice.J Cell Biol. 2002; 156: 1099-1111Crossref PubMed Scopus (1230) Google Scholar whose CLDN1 knockout mouse established the importance of epidermal TJ and CLDN1. These mice died within 24 hours of birth with wrinkled skin, severe dehydration, and increased epidermal permeability as measured by dye studies and transepidermal water loss.7Furuse M. Hata M. Furuse K. Yoshida Y. Haratake A. Sugitani Y. et al.Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice.J Cell Biol. 2002; 156: 1099-1111Crossref PubMed Scopus (1230) Google Scholar In our AD studies, we also observed an inverse correlation between CLDN1 expression and markers of TH2 polarity (total eosinophil counts and serum total IgE).6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar This has led us to speculate that TJ defects may promote greater TH2 polarity, which is a characteristic of subjects with EH.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar We hypothesized that TJ defects such as those we observed in subjects with AD may promote HSV-1 infections on that basis. In addition, viruses often commandeer intercellular junction proteins as a means of viral entry.5Niessen C.M. Tight junctions/adherens junctions: basic structure and function.J Invest Dermatol. 2007; 127: 2525-2532Crossref PubMed Scopus (508) Google Scholar For example, wild-type HSV-1 infects keratinocytes through a complex interaction involving the viral envelope glycoprotein gD with either nectin-1 (PVRL1) or HSV entry mediator.8Gonzalez-Mariscal L. Garay E. Lechuga S. Virus interaction with the apical junctional complex.Front Biosci. 2009; 14: 731-768Crossref Scopus (28) Google Scholar, 9Geraghty R.J. Krummenacher C. Cohen G.H. Eisenberg R.J. Spear P.G. Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.Science. 1998; 280: 1618-1620Crossref PubMed Scopus (784) Google Scholar Nectin-1, a Ca2Boguniewicz M. Leung D.Y. Recent insights into atopic dermatitis and implications for management of infectious complications.J Allergy Clin Immunol. 2010; 125: 4-13Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar-independent cell adhesion protein of the immunoglobulin superfamily, colocalizes with E-cadherin and β-catenin to form another intercellular junctional complex called the adherens junction. Previous studies have shown that the susceptibility of human keratinocytes to HSV-1 infection is inversely related to the degree of cell-cell contact and confluency.9Geraghty R.J. Krummenacher C. Cohen G.H. Eisenberg R.J. Spear P.G. Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor.Science. 1998; 280: 1618-1620Crossref PubMed Scopus (784) Google Scholar, 10Schelhaas M. Jansen M. Haase I. Knebel-Morsdorf D. Herpes simplex virus type 1 exhibits a tropism for basal entry in polarized epithelial cells.J Gen Virol. 2003; 84: 2473-2484Crossref PubMed Scopus (51) Google Scholar This suggests that healthy junctional complexes may be a key deterrent to the spread of HSV-1 from one keratinocyte to another. In these studies, we tested the hypothesis that the silencing of epidermal CLDN1 would enhance HSV-1 infectivity of human keratinocytes. We had previously demonstrated that reductions in CLDN1 adversely affected measures of TJ integrity.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Primary human keratinocytes (PHKs) were isolated from foreskin as previously described6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar and transiently transfected with scrambled (control) or CLDN1 (100 nmol/L) small interfering RNA (siRNA; Santa Cruz Biotechnology, Santa Cruz, Calif) by using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, Calif; see this article’s complete Methods in the Online Repository at www.jacionline.org). We have previously demonstrated that this knockdown approach led to a >50% reduction in claudin-1 transcripts and adversely affected TJ function as demonstrated by a 50% reduction in transepithelial electrical resistance and doubling of the 50% permeability.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Importantly, this level of CLDN1 reduction mirrored what we had observed in AD samples. Twenty-four to 48 hours posttransfection, PHKs were differentiated in Dulbecco modified Eagle medium (DMEM) with 10% heat-inactivated FBS (HI-FBS; Invitrogen/Gibco) for 24 hours and subsequently infected with the highly virulent HSV-1 strain F (provided by Dr D. C. Johnson, Oregon Health and Science University) at a multiplicity of infection of 0.1 (see this article’s complete Methods in the Online Repository). The multiplicity of infection was chosen based on findings from viral titration (0.01-10 multiplicity of infection) studies on differentiated and undifferentiated PHKs, respectively, used as negative and positive controls (data not shown). After 2 hours, the viral inoculum was removed, and PHKs were cultured for 24 hours in DMEM containing 5% HI-FBS and 0.4% human-γ-globulin (0.5 mg/mL; Sigma, St Louis, Mo) to neutralize extracellular virus. HSV-infected PHKs were fixed in 4% formaldehyde and stained with a polyclonal rabbit anti–HSV-1 (1:500; Dako, Carpinteria, Calif) antibody followed by Alexa Fluor 488 donkey-antirabbit IgG H+L (1:1000; Invitrogen) and 4’,6-diamidino-2-phenyl-indole, dihydrochloride (DAPI; 1:10,000; Invitrogen). For each sample, 6 random fields were captured at identical acquisition settings and analyzed computationally to quantify differences objectively in focal forming units (FFUs; see complete Methods in the Online Repository). A FFU was defined as a cluster of 3 or more adjacent HSV-1–positive cells. CLDN1 depletion significantly increased the number and size of HSV-1 FFUs (Fig 1, A) compared with scrambled siRNA–transfected PHK (Fig 1, B). An average of 4.8 ± 0.7 FFUs/field were counted in CLDN1 knockdown PHKs, whereas only 2.5 ± 0.5 FFUs/field were detected in the control cells (P = .05; n = 6; Fig 1, C). The diameters of FFUs were significantly greater in keratinocytes whose CLDN1 expression was reduced (207 ± 22 μm) compared with controls (152 ± 40 μm; P = .03; n = 6; Fig 1, D). Similar findings were observed when evaluating FFU area (P = .04; n = 6; Fig 1, E) in PHKs after CLDN1 knockdown (15,649 ± 4367 pixels) compared with control transfection (9902 ± 4943 pixels). The increased frequency of HSV-1–infected cells in CLDN1 knockdown PHKs was confirmed by an infectious center assay (see this article’s complete Methods in the Online Repository). More CLDN1 siRNA–transfected PHKs were infected with HSV-1 (38 ± 6%; n = 3) than control PHKs (28 ± 7%; n = 3; P = .003; see this article’s Fig E1 in the Online Repository at www.jacionline.org). Importantly, the observed increase in HSV-1 infectivity in CLDN1 knockdown PHKs could not be explained by reduced expression of nectin-1, the HSV-1 receptor. Indeed, nectin-1 mRNA expression was unaffected by CLDN1 silencing (n = 5; see this article’s Fig E2 in the Online Repository at www.jacionline.org). However, we cannot rule out the possibility that reductions in CLDN1 and the effects on TJ function might reduce nectin-1 membrane localization. It is also important to highlight that our in vitro model is sufficient to induce functional TJ as demonstrated by enhanced transepithelial resistance and reduced permeability.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar However, it is not enough to induce terminal differentiation (eg, filaggrin expression and cornified envelope). Although this might be a limitation of the methodology, we believe this condition well reflects the in vivo situation. In AD subjects with history of EH (ADEH+), often the virus spreading is a result of reactivation of a silent infection (inside-out), suggesting that the first barrier structure the virus will encounter is indeed the TJ (located in the stratum granulosum, just below the SC). As part of the National Institute of Allergy and Infectious Diseases–funded Atopic Dermatitis and Vaccinia Network (ADVN; reviewed in a previous publication by Beck et al1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), we performed a preliminary study to evaluate whether CLDN1 polymorphisms were associated with EH in AD subjects. To do this, we used a haplotype-tagging SNP approach with genetic markers available in the public arena (n = 132 in single nucleotide polymorphism database) in 414 European Americans and 328 African Americans (see this article’s complete Methods and Table E1 in the Online Repository at www.jacionline.org). We previously reported modest associations (P = .003-.05) between SNP variants throughout the CLDN1 gene and AD that were more significant among African Americans.6De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar In this study, we addressed whether genetic variations in CLDN1 contribute to risk of EH in both North American AD populations. The EH subphenotype was defined as subjects with AD who had at least 1 EH episode documented by an ADVN investigator (or a physician affiliated with the same academic center) or diagnosed clinically by an outside physician where the HSV infection was confirmed by PCR, tissue immunofluorescence, Tzanck smear, and/or culture. The study was approved by the institutional review boards at National Jewish Health, Johns Hopkins University, Oregon Health and Science University, the University of California San Diego, Children’s Hospital of Boston, and the University of Rochester Medical Center. All subjects gave written informed consent before participation. To determine whether SNPs in CLDN1 were associated with EH, the Cochran-Armitage trend test using the PLINK software (http://pngu.mgh.harford.edu/∼purcell/plink/) was performed within each AD population and confirmed with the adaptive permutation test. We observed only modest associations between CLDN1 SNPs and EH in our North American AD populations. We wondered whether the strong association we observed between both filaggrin (FLG) null mutations R501X and 2282del4 and EH (odds ratio [OR], 10.1 [value range: 4.7-22.1]; P = 1.99 × 1011Shimazaki J. Higa K. Kato N. Satake Y. Barrier function of cultivated limbal and oral mucosal epithelial cell sheets.Invest Ophthalmol Vis Sci. 2009; 50: 5672-5680Crossref PubMed Scopus (27) Google Scholar) was masking a lesser effect from CLDN1 variants.4Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 507-513Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar To address this, we excluded subjects who had 1 or both of these FLG mutations and reanalyzed the associations with all CLDN1 SNPs. In the European American group, an intronic SNP (rs3774032; OR, 0.59 [0.35-0.98]; P = .037) was marginally associated with EH (Fig 2) but not significantly after adaptive permutations adjustment (P = .10). When excluding subjects with a FLG mutation (n = 29 [36.3%]; Table I), the association became slightly more significant (OR, 0.44 [0.22-0.85]; P = .0225) and still significant after adaptive permutations adjustment (P = .035). Interestingly, 1 additional intronic SNP emerged (rs3732923; OR, 1.93 [1.21-3.07]; P = .0010; adaptive permutation, P = .0005; Fig 2). Although the EH sample size in the African American population was too small (n = 21) for meaningful analyses, a SNP in the promoter region was associated with EH (rs3954259; OR, 2.16 [1.02-4.59]; P = .040; adaptive permutation, P = .033). Limitations of these studies are the small sample size and the superficial coverage of CLDN1 loci. Nevertheless, these findings implicate CLDN1 mutations in susceptibility to widespread HSV skin infections in subjects with AD, especially those who do not have a FLG mutation.Table IADVN registry participant characteristicsCharacteristicEuropean AmericanAfrican AmericanADEH+ADEH–ADEH+ADEH–Sample size9316521155Males, n (%)49 (52.7)47 (28.5)8 (38.1)35 (22.6)Age (y), mean (SD)22.9 (21.5)38.0 (14.5)22.8 (19.0)36.4 (11.0)AD onset <5 y, n (%)77 (97.5)101 (78.3)14 (93.3)77 (68.1)IgE level, median (interquartile range)*Total serum IgE levels were significantly higher in patients with ADEH+ compared with both patients with ADEH– and nonatopic, healthy controls even after adjusting for age (P < .001).2225 (611-9034)252 (85-1275)3505 (571-9429)425 (129-1194)EASI,†EASI was determined by the percentage of eczema area on a 7-point ordinal scale: 0, no eruption; 1, <10%; 2, 10% to 29%; 3, 30% to 49%; 4, 50% to 69%; 5, 70% to 89%; and 6, 90% to 100%. median (interquartile range)‡EASI was significantly higher among patients with ADEH+ compared with patients with ADEH– in both racial groups even after adjusting for age (P < .001).10.0 (4.4-15.8)3.2 (1.3-8.4)10.9 (6.8-15.8)3.0 (1.5-7.4)FLG null allele carrier, n (%)29 (36.3)36 (22.1)2 (12.5)9 (6.0)ADEH+, AD with a history of eczema herpeticum; ADEH–, AD without a history of EH; EASI, Eczema Area and Severity Index.∗ Total serum IgE levels were significantly higher in patients with ADEH+ compared with both patients with ADEH– and nonatopic, healthy controls even after adjusting for age (P < .001).† EASI was determined by the percentage of eczema area on a 7-point ordinal scale: 0, no eruption; 1, <10%; 2, 10% to 29%; 3, 30% to 49%; 4, 50% to 69%; 5, 70% to 89%; and 6, 90% to 100%.‡ EASI was significantly higher among patients with ADEH+ compared with patients with ADEH– in both racial groups even after adjusting for age (P < .001). Open table in a new tab ADEH+, AD with a history of eczema herpeticum; ADEH–, AD without a history of EH; EASI, Eczema Area and Severity Index. In conclusion, this study and previously reported work implicate both SC and TJ epidermal barrier defects as a mechanism to explain the susceptibility of subjects with AD to widespread cutaneous infections with HSV and possibly other viral pathogens.4Gao P.S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 507-513Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar In support of this notion, our patients with EH more commonly experienced infections with molluscum contagiosum than subjects with AD with no history of EH (29% vs 2%, respectively).1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar More of our subjects with EH (16%) had recurrent HSV keratitis compared with only 1% of subjects with AD with no history of EH.1Beck L.A. Boguniewicz M. Hata T. Schneider L.C. Hanifin J. Gallo R. et al.Phenotype of atopic dermatitis subjects with a history of eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 260-269Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar It is possible that the same mechanism may responsible for their ocular disease because corneal epithelium possesses TJs and these structures express claudin-1.11Shimazaki J. Higa K. Kato N. Satake Y. Barrier function of cultivated limbal and oral mucosal epithelial cell sheets.Invest Ophthalmol Vis Sci. 2009; 50: 5672-5680Crossref PubMed Scopus (27) Google Scholar, 12Yoshida Y. Ban Y. Kinoshita S. Tight junction transmembrane protein claudin subtype expression and distribution in human corneal and conjunctival epithelium.Invest Ophthalmol Vis Sci. 2009; 50: 2103-2108Crossref PubMed Scopus (64) Google Scholar In this study, we show both mechanistic and genetic results that implicate TJs as a critical barrier structure important in containing the spread of epidermal HSV-1 infections. Defects in TJ compared with the SC may be particularly relevant in viral infections in which the virus enters from a basolateral direction, as is the case with HSV reactivation. Collectively, this work highlights a previously underappreciated role for TJ proteins in cutaneous host defense and may lead to new therapeutic strategies. We acknowledge Melissa Dubois, PhD, and Todd W. Wisner at Oregon Health and Science University (Beaverton, Ore) for helpful suggestions on HSV-1 experiments. Jonathan Rebhahn, MS, at the University of Rochester Medical Center (Rochester, NY) developed the software to quantify FFUs. Mary Bolognino, BS, at the University of Rochester Medical Center (Rochester, NY) helped with PHK studies. We also acknowledge several groups whose efforts made the clinical enrollment possible: ADVN coordinators (Patricia Taylor, NP; Trista Berry, BS; Susan Tofte, FNP; Shahana Baig-Lewis, MPH; Peter Brown, BS; Lisa Heughan, BA, CCRC; Meggie Nguyen, BS; Doru Alexandrescu, MD; Lorianne Stubbs, RC; Reena Vaid, MD; Diana Lee, MD), ADVN regulatory advisors (Judy Lairsmith, RN), biological sample tracking (Johns Hopkins University, Tracey Hand, MSc, Jessica Scarpola; University of Rochester Medical Center, Mary Bolognino, MS), National Institute of Allergy and Infectious Diseases–Division of Allergy, Immunology and Transplantation oversight (Marshall Plaut, MD, and Joy Laurienzo Panza, RN, BSN), Rho, Inc, statistical support (Daniel Zaccaro, MS; Brian Armstrong, MPH) Rho, Inc, general study support and oversight (Jamie Reese, BS; Susi Lieff, PhD; Gloria David, PhD, MHSc), and all the patients who participated in this study. Human keratinocytes (PHKs) were isolated from neonatal foreskin.E1De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar PHKs were cultured in keratinocyte-SFM (Invitrogen/Gibco, Carlsbad, Calif) with 1% Pen/Strep, 0.2% amphotericin B (Invitrogen/Gibco). To differentiate PHK, cells were grown in DMEM (Invitrogen/Gibco) with 10% HI-FBS (Invitrogen/Gibco) and 1% Pen/Strep, 0.2% amphotericin B (Invitrogen/Gibco). Primary human keratinocytes were plated on glass coverslips at 2 to 3 × 105 cells/well in a 6-well plate or at 2 to 3 × 104 cells/filter in Transwell inserts (Costar; Corning Inc, Corning, New York; Polyester Membrane, 0.4 μm pore size, 6.5 mm insert) in keratinocyte-SFM without antibiotics. The day after plating, cells were transfected with claudin-1 specific or control (scrambled) siRNAs (Santa Cruz Biotechnology, Santa Cruz, Calif) by using Lipofectamine 2000 Transfection Reagent (Invitrogen). Infection studies were performed by using the highly virulent HSV-1 strain F (provided by Dr D. C. Johnson). Twenty-four to 48 hours posttransfection with 100 nmol/L claudin-1 or control siRNA, the cells were differentiated in DMEM with 10% heat-inactivated FBS for 24 hours. Cells were washed twice with HBSS and infected with HSV-1 strain F at a multiplicity of infection of 0.1 in DMEM containing 1% heat-inactivated FBS at 37°C, with rocking every 15 minutes. After 2 hours, the viral inoculum was removed, and the cells were washed 2 times with HBSS and incubated in DMEM containing 5% HI-FBS and 0.4% human-γ-globulin (Sigma; final concentration, 0.5 mg/mL) for 24 hours to neutralize any extracellular virus. It is important to note that our PHKs are grown under differentiating conditions that induce functional TJs as demonstrated by enhanced transepithelial resistance and reduced permeability,E1De Benedetto A. Rafaels N.M. McGirt L.Y. Ivanov A.I. Georas S.N. Cheadle C. et al.Tight junctions defects in patients with atopic dermatitis.J Allergy Clin Immunol. 2011; 127 (e7): 773-786Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar but they do not fully differentiate, as evidenced by the lack of filaggrin expression. Herpes simplex virus–infected PHKs were washed 2 times with PBS and fixed in 4% formaldehyde/PBS for 20 minutes at room temperature. A polyclonal rabbit anti–HSV-1 (Dako) antibody diluted 1:500 in PBS/1% BSA was placed on the PHK for 1 hour at 37°C followed by Alexa Fluor 488 donkey-antirabbit IgG H+L (1:1000; Invitrogen) and DAPI (1:10,000; Invitrogen). Coverslips were mounted onto slides with SlowFade (Invitrogen). For each sample, 6 random fields were captured at identical acquisition settings. Images were stored in Portable Network Graphics format and analyzed computationally to quantify differences objectively in FFUs. HSV-1 infected cells were assigned to the green channel. Cell density was calculated by counting the number of DAPI-labeled nuclei, assigned to the blue channel. Images were analyzed by using MATLAB (MathWorks, Inc, Natick, MA) to enumerate FFUs, and total cell number. The following FFU measurements were also taken: major axis length (μm), area (μm2), and pixel area (px). The infected cell channel (green) was converted to a binary image using the Otsu method for threshold determination.E2Otsu N. A threshold selection method from gray-level histograms.IEEE Transactions on Systems, Man, and Cybernetics. 1979; 9: 62-66Crossref Google Scholar A morphologic closing (dilation followed by erosion) was performed to reduce noise and fuse individual infected cells into colonies. A distance transform was computed from the binary image, followed by a watershed transform. The colony binary image was then multiplied by its watershed image, the result was dilated slightly, and its perimeter was drawn. Only cells with sizes greater than a predefined threshold were included for further analysis. The infected cell channel (green) was filtered to reduce effects of noise. The resulting image was inverted, followed by a watershed transform. The infected cell watershed image was then multiplied by the colony watershed image to identify cells within previously identified colonies. Finally, a perimeter was drawn around cell borders. Twenty-four hours postinfection, PHKs were treated with 0.05% trypsin-EDTA (Invitrogen/Gibco) for 5 minutes at 37°C followed by gently scraping to lift the cells, washed 3 times in DMEM containing 1% HI-FBS, and vortexed for 30 seconds to disrupt cell clumps. Live cells were counted by using Trypan blue exclusion, and scalar PHK dilutions (1000 to 1 cells in 1 mL media) were plated onto pregrown monolayers of Vero cells in 6-well plates. After 2 hours, 1 mL DMEM containing 5% HI-FBS and 0.4% human-γ-globulin (Sigma; final concentration, 0.5 mg/mL) was added to each well. After 3 days of incubation at 37°C, cells were fixed (75% methanol/25% acetic acid) and stained with 0.1% crystal violet. Plaques were counted by 2 independent investigators, and results were expressed as a percentage of infected PHKs. Reverse transcription was performed from total RNA by using the iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer’s protocol. Quantitative PCR (eg, real-time PCR) was performed by using the iQ SYBER Green Supermix assay system from Bio-Rad Laboratories. All PCR amplifications were carried out in triplicate on an iQ5 Multicolor real-time PCR detection system (Bio-Rad). Primers were designed and synthesized by Integrated DNA Technologies. Primers used in the study were as follows: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward: GAA GGT GAA GGT CGG AGT C, and reverse: GAA GAT GGT GAT GGG ATT TC), CLDN1 (forward: CGA TGA GGT GCA GAA GAT GA, and reverse: CCA GTG AAG AGA GCC TGA CC), and PVRL1 (nectin-1; forward: AGC CAT TAA GGA GAA ACG A, and reverse: TTC CCA ATT TCT CTG CTC T). Relativ

The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine central to the response to endotoxemia, is a putative biomarker in acute lung injury (ALI). To explore MIF as a molecular target and candidate gene in ALI, the MIF gene and protein expression were examined in murine and canine models of ALI (high tidal volume mechanical ventilation, endotoxin exposure) and in patients with either sepsis or sepsis-induced ALI. MIF gene expression and protein levels were significantly increased in each ALI model, with serum MIF levels significantly higher in patients with either sepsis or ALI compared with healthy controls (African- and European-descent). The association of 8 MIF gene polymorphisms [single-nucleotide polymorphisms (SNPs)] (within a 9.7-kb interval on chromosome 22q11.23) with the development of sepsis and ALI in European-descent and African-descent populations was studied next. Genotyping in 506 DNA samples (sepsis patients, sepsis-associated ALI patients, and healthy controls) revealed haplotypes located in the 3′ end of the MIF gene, but not individual SNPs, associated with sepsis and ALI in both populations. These data, generated via functional genomic and genetic approaches, suggest that MIF is a relevant molecular target in ALI. The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine central to the response to endotoxemia, is a putative biomarker in acute lung injury (ALI). To explore MIF as a molecular target and candidate gene in ALI, the MIF gene and protein expression were examined in murine and canine models of ALI (high tidal volume mechanical ventilation, endotoxin exposure) and in patients with either sepsis or sepsis-induced ALI. MIF gene expression and protein levels were significantly increased in each ALI model, with serum MIF levels significantly higher in patients with either sepsis or ALI compared with healthy controls (African- and European-descent). The association of 8 MIF gene polymorphisms [single-nucleotide polymorphisms (SNPs)] (within a 9.7-kb interval on chromosome 22q11.23) with the development of sepsis and ALI in European-descent and African-descent populations was studied next. Genotyping in 506 DNA samples (sepsis patients, sepsis-associated ALI patients, and healthy controls) revealed haplotypes located in the 3′ end of the MIF gene, but not individual SNPs, associated with sepsis and ALI in both populations. These data, generated via functional genomic and genetic approaches, suggest that MIF is a relevant molecular target in ALI.

We hypothesize that pulmonary arterial hypertension (PAH)-associated genes identified by expression profiling of peripheral blood mononuclear cells (PBMCs) from patients with idiopathic pulmonary arterial hypertension (IPAH) can also be identified in PBMCs from scleroderma patients with PAH (PAH-SSc). Gene expression profiles of PBMCs collected from IPAH (n = 9), PAH-SSc (n = 10) patients, and healthy controls (n = 5) were generated using HG_U133A_2.0 GeneChips and were processed by the RMA/GCOS_1.4/SAM_1.21 data analysis pipeline. Disease severity in consecutive patients was assessed by functional status and hemodynamic measurements. The expression profiles were analyzed using PAH severity-stratification, and identified candidate genes were validated with real-time polymerase chain reaction (PCR). Transcriptomics of PBMCs from IPAH patients was highly comparable with that of PMBCs from PAH-SSc patients. The PBMC gene expression patterns significantly correlate with right atrium pressure (RA) and cardiac index (CI), which are known predictors of survival in PAH. Array stratification by RA and CI identified 364 PAH-associated candidate genes. Gene ontology (GO) analysis revealed significant (Z(score) > 1.96) alterations in angiogenesis genes according to PAH severity: matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF) were significantly upregulated in mild as compared with severe PAH and healthy controls, as confirmed by real-time PCR. These data demonstrate that PBMCs from patients with PAH-SSc carry distinct transcriptional expression. Furthermore, our findings suggest an association between angiogenesis-related gene expression and severity of PAH in PAH-SSc patients. Deciphering the role of genes involved in vascular remodeling and PAH development may reveal new treatment targets for this devastating disorder.

Long-chain polyunsaturated fatty acids (PUFA) orchestrate immunity and inflammation through their capacity to be converted to potent inflammatory mediators. We assessed associations of FADS gene cluster polymorphisms and fasting serum PUFA concentrations in a fully ascertained, geographically isolated founder population of European descent. Concentrations of 22 PUFAs were determined by gas chromatography, of which ten fatty acids and five ratios defining FADS1 and FADS2 activity were tested for genetic association against 16 single nucleotide polymorphisms (SNP) in 224 individuals. A cluster of SNPs in tight linkage disequilibrium in the FADS1 gene (rs174537, rs174545, rs174546, rs174553, rs174556, rs174561, rs174568, and rs99780) were strongly associated with arachidonic acid (AA) (P = 5.8 x 10(-7) - 1.7 x 10(-8)) among other PUFAs, but the strongest associations were with the ratio measuring FADS1 activity in the omega-6 series (P = 2.11 x 10(-13) - 1.8 x 10(-20)). The minor allele across all SNPs was consistently associated with decreased omega-6 PUFAs, with the exception of dihomo-gamma-linoleic acid (DHGLA), where the minor allele was consistently associated with increased levels. Our findings in a geographically isolated population with a homogenous dietary environment suggest that variants in the Delta-5 desaturase enzymatic step likely regulate the efficiency of conversion of medium-chain PUFAs to potentially inflammatory PUFAs, such as AA.

<h2>Summary</h2> Atopic dermatitis (AD) is a chronic inflammatory skin disease. Here, we show that phospholipase C-β3 (PLC-β3)-deficient mice spontaneously develop AD-like skin lesions and more severe allergen-induced dermatitis than wild-type mice. Mast cells were required for both AD models and remarkably increased in the skin of <i>Plcb3</i>^−/− mice because of the increased Stat5 and reduced SHP-1 activities. Mast cell-specific deletion of <i>Stat5</i> gene ameliorated allergen-induced dermatitis, whereas that of <i>Shp1</i> gene encoding Stat5-inactivating SHP-1 exacerbated it. PLC-β3 regulates the expression of periostin in fibroblasts and TSLP in keratinocytes, two proteins critically involved in AD pathogenesis. Furthermore, polymorphisms in <i>PLCB3</i>, <i>SHP1</i>, <i>STAT5A</i>, and <i>STAT5B</i> genes were associated with human AD. Mast cell expression of PLC-β3 was inversely correlated with that of phospho-STAT5, and increased mast cells with high levels of phospho-STAT5 were found in lesional skin of some AD patients. Therefore, STAT5 regulatory mechanisms in mast cells are important for AD pathogenesis.

<h3>Context</h3>Recent studies have shown an association between cigarettes per day (CPD) and a nonsynonymous single-nucleotide polymorphism in CHRNA5, rs16969968.<h3>Objective</h3>To determine whether the association between rs16969968 and smoking is modified by age at onset of regular smoking.<h3>Data Sources</h3>Primary data.<h3>Study Selection</h3>Available genetic studies containing measures of CPD and the genotype of rs16969968 or its proxy.<h3>Data Extraction</h3>Uniform statistical analysis scripts were run locally. Starting with 94 050 ever-smokers from 43 studies, we extracted the heavy smokers (CPD &gt;20) and light smokers (CPD ≤10) with age-at-onset information, reducing the sample size to 33 348. Each study was stratified into early-onset smokers (age at onset ≤16 years) and late-onset smokers (age at onset &gt;16 years), and a logistic regression of heavy vs light smoking with the rs16969968 genotype was computed for each stratum. Meta-analysis was performed within each age-at-onset stratum.<h3>Data Synthesis</h3>Individuals with 1 risk allele at rs16969968 who were early-onset smokers were significantly more likely to be heavy smokers in adulthood (odds ratio [OR] = 1.45; 95% CI, 1.36-1.55; n = 13 843) than were carriers of the risk allele who were late-onset smokers (OR = 1.27; 95% CI, 1.21-1.33, n = 19 505) (P = .01).<h3>Conclusion</h3>These results highlight an increased genetic vulnerability to smoking in early-onset smokers.

Acute Lung Injury (ALI) is a syndrome with high associated mortality characterized by severe hypoxemia and pulmonary infiltrates in patients with critical illness. We conducted the first investigation to use the genome wide association (GWA) approach to identify putative risk variants for ALI. Genome wide genotyping was performed using the Illumina Human Quad 610 BeadChip. We performed a two-stage GWA study followed by a third stage of functional characterization. In the discovery phase (Phase 1), we compared 600 European American trauma-associated ALI cases with 2266 European American population-based controls. We carried forward the top 1% of single nucleotide polymorphisms (SNPs) at p<0.01 to a replication phase (Phase 2) comprised of a nested case-control design sample of 212 trauma-associated ALI cases and 283 at-risk trauma non-ALI controls from ongoing cohort studies. SNPs that replicated at the 0.05 level in Phase 2 were subject to functional validation (Phase 3) using expression quantitative trait loci (eQTL) analyses in stimulated B-lymphoblastoid cell lines (B-LCL) in family trios. 159 SNPs from the discovery phase replicated in Phase 2, including loci with prior evidence for a role in ALI pathogenesis. Functional evaluation of these replicated SNPs revealed rs471931 on 11q13.3 to exert a cis-regulatory effect on mRNA expression in the PPFIA1 gene (p = 0.0021). PPFIA1 encodes liprin alpha, a protein involved in cell adhesion, integrin expression, and cell-matrix interactions. This study supports the feasibility of future multi-center GWA investigations of ALI risk, and identifies PPFIA1 as a potential functional candidate ALI risk gene for future research.

Asthma is a complex disease with sex-specific differences in prevalence. Candidate gene studies have suggested that genotype-by-sex interaction effects on asthma risk exist, but this has not yet been explored at a genome-wide level. We aimed to identify sex-specific asthma risk alleles by performing a genome-wide scan for genotype-by-sex interactions in the ethnically diverse participants in the EVE Asthma Genetics Consortium. We performed male- and female-specific genome-wide association studies in 2653 male asthma cases, 2566 female asthma cases and 3830 non-asthma controls from European American, African American, African Caribbean and Latino populations. Association tests were conducted in each study sample, and the results were combined in ancestry-specific and cross-ancestry meta-analyses. Six sex-specific asthma risk loci had P-values < 1 × 10−6, of which two were male specific and four were female specific; all were ancestry specific. The most significant sex-specific association in European Americans was at the interferon regulatory factor 1 (IRF1) locus on 5q31.1. We also identify a Latino female-specific association in RAP1GAP2. Both of these loci included single-nucleotide polymorphisms that are known expression quantitative trait loci and have been associated with asthma in independent studies. The IRF1 locus is a strong candidate region for male-specific asthma susceptibility due to the association and validation we demonstrate here, the known role of IRF1 in asthma-relevant immune pathways and prior reports of sex-specific differences in interferon responses.

<h3>Background</h3> COPD is a heterogeneous disease, but there is little consensus on specific definitions for COPD subtypes. Unsupervised clustering offers the promise of 'unbiased' data-driven assessment of COPD heterogeneity. Multiple groups have identified COPD subtypes using cluster analysis, but there has been no systematic assessment of the reproducibility of these subtypes. <h3>Objective</h3> We performed clustering analyses across 10 cohorts in North America and Europe in order to assess the reproducibility of (1) correlation patterns of key COPD-related clinical characteristics and (2) clustering results. <h3>Methods</h3> We studied 17 146 individuals with COPD using identical methods and common COPD-related characteristics across cohorts (FEV₁, FEV₁/FVC, FVC, body mass index, Modified Medical Research Council score, asthma and cardiovascular comorbid disease). Correlation patterns between these clinical characteristics were assessed by principal components analysis (PCA). Cluster analysis was performed using k-medoids and hierarchical clustering, and concordance of clustering solutions was quantified with normalised mutual information (NMI), a metric that ranges from 0 to 1 with higher values indicating greater concordance. <h3>Results</h3> The reproducibility of COPD clustering subtypes across studies was modest (median NMI range 0.17–0.43). For methods that excluded individuals that did not clearly belong to any cluster, agreement was better but still suboptimal (median NMI range 0.32–0.60). Continuous representations of COPD clinical characteristics derived from PCA were much more consistent across studies. <h3>Conclusions</h3> Identical clustering analyses across multiple COPD cohorts showed modest reproducibility. COPD heterogeneity is better characterised by continuous disease traits coexisting in varying degrees within the same individual, rather than by mutually exclusive COPD subtypes.

Abstract Common variants at many loci have been robustly associated with asthma but explain little of the overall genetic risk. Here we investigate the role of rare (&lt;1%) and low-frequency (1–5%) variants using the Illumina HumanExome BeadChip array in 4,794 asthma cases, 4,707 non-asthmatic controls and 590 case–parent trios representing European Americans, African Americans/African Caribbeans and Latinos. Our study reveals one low-frequency missense mutation in the GRASP gene that is associated with asthma in the Latino sample ( P =4.31 × 10 −6 ; OR=1.25; MAF=1.21%) and two genes harbouring functional variants that are associated with asthma in a gene-based analysis: GSDMB at the 17q12–21 asthma locus in the Latino and combined samples ( P =7.81 × 10 −8 and 4.09 × 10 −8 , respectively) and MTHFR in the African ancestry sample ( P =1.72 × 10 −6 ). Our results suggest that associations with rare and low-frequency variants are ethnic specific and not likely to explain a significant proportion of the ‘missing heritability’ of asthma.

Genome-wide association studies (GWAS) have been employed in the field of allergic disease, and significant associations have been published for nearly 100 asthma genes/loci. An outcome of GWAS in allergic disease has been the formation of national and international collaborations leading to consortia meta-analyses, and an appreciation for the specificity of genetic associations to sub-phenotypes of allergic disease. Molecular genetics has undergone a technological revolution, leading to next-generation sequencing strategies that are increasingly employed to hone in on the causal variants associated with allergic diseases. Unmet needs include the inclusion of diverse cohorts and strategies for managing big data.

Abstract Pharmaceutical drugs targeting dyslipidemia and cardiovascular disease (CVD) may increase the risk of fatty liver disease and other metabolic disorders. To identify potential novel CVD drug targets without these adverse effects, we perform genome-wide analyses of participants in the HUNT Study in Norway (n = 69,479) to search for protein-altering variants with beneficial impact on quantitative blood traits related to cardiovascular disease, but without detrimental impact on liver function. We identify 76 (11 previously unreported) presumed causal protein-altering variants associated with one or more CVD- or liver-related blood traits. Nine of the variants are predicted to result in loss-of-function of the protein. This includes ZNF529 :p.K405X, which is associated with decreased low-density-lipoprotein (LDL) cholesterol (P = 1.3 × 10 −8 ) without being associated with liver enzymes or non-fasting blood glucose. Silencing of ZNF529 in human hepatoma cells results in upregulation of LDL receptor and increased LDL uptake in the cells. This suggests that inhibition of ZNF529 or its gene product should be prioritized as a novel candidate drug target for treating dyslipidemia and associated CVD.

In recent years, the genomics community has witnessed the growth of large research biobanks, which collect DNA samples for research purposes. Depending on how and where the samples are genotyped, biobanks also offer the potential opportunity to return actionable genomic results to the clinical setting. We developed a preemptive clinical pharmacogenomic implementation initiative via a health system-wide research biobank at the University of Colorado. Here, we describe how preemptive return of clinical pharmacogenomic results via a research biobank is feasible, particularly when coupled with strong institutional support to maximize the impact and efficiency of biobank resources, a multidisciplinary implementation team, automated clinical decision support tools, and proactive strategies to engage stakeholders early in the clinical decision support tool development process.

Platelet aggregation at the site of atherosclerotic vascular injury is the underlying pathophysiology of myocardial infarction and stroke. To build upon prior GWAS, here we report on 16 loci identified through a whole genome sequencing (WGS) approach in 3,855 NHLBI Trans-Omics for Precision Medicine (TOPMed) participants deeply phenotyped for platelet aggregation. We identify the RGS18 locus, which encodes a myeloerythroid lineage-specific regulator of G-protein signaling that co-localizes with expression quantitative trait loci (eQTL) signatures for RGS18 expression in platelets. Gene-based approaches implicate the SVEP1 gene, a known contributor of coronary artery disease risk. Sentinel variants at RGS18 and PEAR1 are associated with thrombosis risk and increased gastrointestinal bleeding risk, respectively. Our WGS findings add to previously identified GWAS loci, provide insights regarding the mechanism(s) by which genetics may influence cardiovascular disease risk, and underscore the importance of rare variant and regulatory approaches to identifying loci contributing to complex phenotypes.

Abstract Integrating genetic information with metabolomics has provided new insights into genes affecting human metabolism. However, gene-metabolite integration has been primarily studied in individuals of European Ancestry, limiting the opportunity to leverage genomic diversity for discovery. In addition, these analyses have principally involved known metabolites, with the majority of the profiled peaks left unannotated. Here, we perform a whole genome association study of 2,291 metabolite peaks (known and unknown features) in 2,466 Black individuals from the Jackson Heart Study. We identify 519 locus-metabolite associations for 427 metabolite peaks and validate our findings in two multi-ethnic cohorts. A significant proportion of these associations are in ancestry specific alleles including findings in APOE, TTR and CD36 . We leverage tandem mass spectrometry to annotate unknown metabolites, providing new insight into hereditary diseases including transthyretin amyloidosis and sickle cell disease. Our integrative omics approach leverages genomic diversity to provide novel insights into diverse cardiometabolic diseases.

Background Atopic dermatitis (AD) is characterized by TH2-dominated skin inflammation and systemic response to cutaneously encountered antigens. The TH2 cytokines IL-4 and IL-13 play a critical role in the pathogenesis of AD. The Q576->R576 polymorphism in the IL-4 receptor alpha (IL-4Rα) chain common to IL-4 and IL-13 receptors alters IL-4 signaling and is associated with asthma severity. Objective We sought to investigate whether the IL-4Rα R576 polymorphism is associated with AD severity and exaggerates allergic skin inflammation in mice. Methods Nighttime itching interfering with sleep, Rajka-Langeland, and Eczema Area and Severity Index scores were used to assess AD severity. Allergic skin inflammation following epicutaneous sensitization of mice 1 or 2 IL-4Rα R576 alleles (QR and RR) and IL-4Rα Q576 (QQ) controls was assessed by flow cytometric analysis of cells and quantitative RT-PCR analysis of cytokines in skin. Results The frequency of nighttime itching in 190 asthmatic inner-city children with AD, as well as Rajka-Langeland and Eczema Area and Severity Index scores in 1116 White patients with AD enrolled in the Atopic Dermatitis Research Network, was higher in subjects with the IL-4Rα R576 polymorphism compared with those without, with statistical significance for the Rajka-Langeland score. Following epicutaneous sensitization of mice with ovalbumin or house dust mite, skin infiltration by CD4+ cells and eosinophils, cutaneous expression of Il4 and Il13, transepidermal water loss, antigen-specific IgE antibody levels, and IL-13 secretion by antigen-stimulated splenocytes were significantly higher in RR and QR mice compared with QQ controls. Bone marrow radiation chimeras demonstrated that both hematopoietic cells and stromal cells contribute to the mutants’ exaggerated allergic skin inflammation. Conclusions The IL-4Rα R576 polymorphism predisposes to more severe AD and increases allergic skin inflammation in mice. Atopic dermatitis (AD) is characterized by TH2-dominated skin inflammation and systemic response to cutaneously encountered antigens. The TH2 cytokines IL-4 and IL-13 play a critical role in the pathogenesis of AD. The Q576->R576 polymorphism in the IL-4 receptor alpha (IL-4Rα) chain common to IL-4 and IL-13 receptors alters IL-4 signaling and is associated with asthma severity. We sought to investigate whether the IL-4Rα R576 polymorphism is associated with AD severity and exaggerates allergic skin inflammation in mice. Nighttime itching interfering with sleep, Rajka-Langeland, and Eczema Area and Severity Index scores were used to assess AD severity. Allergic skin inflammation following epicutaneous sensitization of mice 1 or 2 IL-4Rα R576 alleles (QR and RR) and IL-4Rα Q576 (QQ) controls was assessed by flow cytometric analysis of cells and quantitative RT-PCR analysis of cytokines in skin. The frequency of nighttime itching in 190 asthmatic inner-city children with AD, as well as Rajka-Langeland and Eczema Area and Severity Index scores in 1116 White patients with AD enrolled in the Atopic Dermatitis Research Network, was higher in subjects with the IL-4Rα R576 polymorphism compared with those without, with statistical significance for the Rajka-Langeland score. Following epicutaneous sensitization of mice with ovalbumin or house dust mite, skin infiltration by CD4+ cells and eosinophils, cutaneous expression of Il4 and Il13, transepidermal water loss, antigen-specific IgE antibody levels, and IL-13 secretion by antigen-stimulated splenocytes were significantly higher in RR and QR mice compared with QQ controls. Bone marrow radiation chimeras demonstrated that both hematopoietic cells and stromal cells contribute to the mutants’ exaggerated allergic skin inflammation. The IL-4Rα R576 polymorphism predisposes to more severe AD and increases allergic skin inflammation in mice.

<h3>Background</h3> Atopic dermatitis (AD) is a chronic inflammatory skin condition with a highly variable clinical phenotype. <h3>Objective</h3> This study aimed to identify historical and clinical features and biomarkers associated with AD severity. <h3>Methods</h3> A US registry of extensively phenotyped AD participants (aged 0.73-80 years) were enrolled at 9 academic centers. Information on family and personal medical history, examination, skin swabs (culture), and serum biomarkers was collected to evaluate their association with AD severity. <h3>Results</h3> Participants with AD (N = 2862) whose disease was categorized as mild (11.6%), moderate (58.0%), or severe (30.4%) based on Rajka-Langeland scoring were enrolled. The trend test, when adjusting for gender, race, and age, demonstrated that severity was strongly (<i>P</i> ≤ .04) associated with a personal/family history of allergic disorders, history of alopecia, exposure to passive smoke, ocular herpes infection, skin bacterial and viral infections, and history of arrhythmia. Features observed more frequently (<i>P</i> ≤ .002), as a function of severity, included skin infections (impetigo, human papillomavirus, and molluscum contagiosum virus), <i>Staphylococcus aureus</i> colonization, excoriations, hyperlinear palms, ichthyosis, blepharitis, conjunctivitis, ectropion, and wheezing. Serum IgE, allergen and food (≤6 years) Phadiatop, and eosinophilia were strongly linked to severity (<i>P</i> < .001). <h3>Conclusions</h3> In a diverse US AD population, severity was associated with a history of atopic disorders, skin and extracutaneous bacterial and viral infections (by history and physical examination), higher IgE, eosinophilia and allergen sensitization, atopic skin manifestations (ie, excoriation, hyperlinear palms, and ichthyosis), and atopic ocular features (ie, blepharitis, conjunctivitis, and ectropion) as well as asthma findings (ie, wheezing). Data from our prospective registry significantly advance our understanding of AD phenotypes and endotypes, which is critical to achieve optimal management.

Group 1 pulmonary arterial hypertension (PAH) is a progressive fatal condition characterized by right ventricular (RV) failure with worse outcomes in connective tissue disease (CTD). Obstructive sleep apnea and sleep-related hypoxia may contribute to RV dysfunction, though the relationship remains unclear. The aim of this study was to prospectively evaluate the association of the apnea-hypopnea index (AHI) and sleep-related hypoxia with RV function and survival. Pulmonary Vascular Disease Phenomics (National Heart, Lung, and Blood Institute) cohort participants (patients with group 1 PAH, comparators, and healthy control participants) with sleep studies were included. Multimodal RV functional measures were examined in association with AHI and percentage of recording time with oxygen saturation <90% (T90) per 10-unit increment. Linear models, adjusted for demographics, oxygen, diffusing capacity of the lungs for carbon monoxide, pulmonary hypertension medications, assessed AHI and T90, and RV measures. Log-rank test/Cox proportional hazards models adjusted for demographics, oxygen, and positive airway pressure were constructed for transplantation-free survival analyses. Analysis included 186 participants with group 1 PAH with a mean age of 52.6 ± 14.1 years; 71.5% were women, 80.8% were Caucasian, and there were 43 events (transplantation or death). AHI and T90 were associated with decreased RV ejection fraction (on magnetic resonance imaging), by 2.18% (−2.18; 95% CI: −4.00 to −0.36; P = 0.019) and 0.93% (−0.93; 95% CI: −1.47 to −0.40; P < 0.001), respectively. T90 was associated with increased RV systolic pressure (on echocardiography), by 2.52 mm Hg (2.52; 95% CI: 1.61 to 3.43; P < 0.001); increased mean pulmonary artery pressure (on right heart catheterization), by 0.27 mm Hg (0.27; 95% CI: 0.05 to 0.49; P = 0.019); and RV hypertrophy (on electrocardiography), 1.24 mm (1.24; 95% CI: 1.10 to 1.40; P < 0.001). T90, but not AHI, was associated with a 17% increased 5-year risk for transplantation or death (HR: 1.17; 95% CI: 1.07 to 1.28). In non-CTD-associated PAH, T90 was associated with a 21% increased risk for transplantation or death (HR: 1.21; 95% CI: 1.08 to 1.34). In CTD-associated PAH, T90 was associated with RV dysfunction, but not death or transplantation. Sleep-related hypoxia was more strongly associated than AHI with measures of RV dysfunction, death, or transplantation overall and in group 1 non-CTD-associated PAH but only with RV dysfunction in CTD-associated PAH. (Pulmonary Vascular Disease Phenomics Program [PVDOMICS]; NCT02980887)

Abstract Why do common diseases such as allergy and asthma have complex patterns of inheritance while other, relatively uncommon diseases approximate simple Mendelian traits? There is good evidence that the dysfunctioning of highly involved, interactive genetic networks is implicated in most complex diseases. Here, Kathleen Barnes and David Marsh review recent studies of asthma, which provide a good model for illustrating this point.

Linkage of asthma and high total serum IgE levels to chromosome 12q15–q24.1 has been recently described. To evaluate this region further in regard to total IgE responsiveness, we genotyped 52 unrelated German children with persistently “high” total serum IgE (selected from a noninterventional prospective multicenter cohort study) and their parents. We carefully defined a most extreme IgE phenotype and analyzed it as a dichotomous trait. We tested for linkage between high total IgE concentrations and nine polymorphic microsatellite markers on chromosome 12q15–q24.1 using the transmission/disequilibrium test. Evidence for linkage and allelic association for high total IgE was observed for four markers in this region. This study demonstrates the value of using extreme phenotypes in genetic analysis of a complex quantitative trait.

Dermatophagoides pteronyssinus (Der p) is one of the most frequently implicated allergens in atopic diseases. Although HLA could play an important role in the development of the IgE response to the Der p allergens, genetic regulation by non-HLA genes influences certain HLA-associated IgE responses to complex allergens.To clarify genetic control for the expression of Der p-specific IgE responsiveness, we conducted a genome-wide search for genes influencing Der p-specific IgE antibody levels by using 45 Caucasian and 53 African American families ascertained as part of the Collaborative Study on the Genetics of Asthma (CSGA).Specific IgE antibody levels to the Der p crude allergen and to the purified allergens Der p 1 and Der p 2 were measured. Multipoint, nonparametric linkage analysis of 370 polymorphic markers was performed with the GENEHUNTER program.The best evidence of genes controlling specific IgE response to Der p was obtained in 2 novel regions: chromosomes 2q21-q23 (P = .0033 for Caucasian subjects) and 8p23-p21 (P = .0011 for African American subjects). Three regions previously proposed as candidate regions for atopy, total IgE, or asthma also showed evidence for linkage to Der p-specific IgE responsiveness: 6p21 (P = .0064) and 13q32-q34 (P = 0.0064) in Caucasian subjects and 5q23-q33 (P = 0.0071) in African American subjects.No single locus generated overwhelming evidence for linkage in terms of established criteria and guidelines for a genome-wide screening, which supports previous assertions of a heterogeneous etiology for Der p-specific IgE responsiveness. Two novel regions, 2q21-q23 and 8p23-p21, that were identified in this study merit additional study.

The gene, CRTH2, encoding a receptor for prostaglandin D(2) (PGD(2)), is located within the peak linkage region for asthma on chromosome (Chr.) 11q reported in African American families. Family-based analysis of asthma and two common SNPs [G1544C and G1651A (rs545659)] in the 3'-untranslated region of CRTH2 showed significant evidence of linkage in the presence of disequilibrium for the 1651G allele (P = 0.003) of SNP rs545659. Haplotype analysis yielded additional evidence of linkage disequilibrium for the 1544G-1651G haplotype (P < 0.001). Population-based case-control analyses were conducted in two independent populations, and demonstrated significant association of the 1544G-1651G haplotype with asthma in an African American population (P = 0.004), and in a population of Chinese children (P < 0.001). Moreover, in the Chinese children the frequency of the 1651G allele in near-fatal asthmatics was significantly higher than mild-to-moderate asthmatics (P = 0.001) and normal controls (P < 0.001). The 1651G allele of SNP re545659 was also associated with a higher degree of bronchial hyperresponsiveness (P < 0.027). Transcriptional pulsing experiments showed that the 1544G-1651G haplotype confers a significantly higher level of reporter mRNA stability, when compared with a non-transmitted haplotype (1544C-1651A), suggesting that the CRTH2 gene on Chr. 11q is a strong candidate gene for asthma.