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1.  Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum 
Background
A subset of atopic dermatitis (AD) is associated with increased susceptibility to eczema herpeticum (ADEH+). We previously reported that common single nucleotide polymorphisms (SNPs) in interferon-gamma (IFNG) and receptor 1 (IFNGR1) were associated with ADEH+ phenotype.
Objective
To interrogate the role of rare variants in IFN-pathway genes for risk of ADEH+.
Methods
We performed targeted sequencing of interferon-pathway genes (IFNG, IFNGR1, IFNAR1 and IL12RB1) in 228 European American (EA) AD patients selected according to their EH status and severity measured by Eczema Area and Severity Index (EASI). Replication genotyping was performed in independent samples of 219 EA and 333 African Americans (AA). Functional investigation of ‘loss-of-function’ variants was conducted using site-directed mutagenesis.
Results
We identified 494 single nucleotide variants (SNVs) encompassing 105kb of sequence, including 145 common, 349 (70.6%) rare (minor allele frequency (MAF) <5%) and 86 (17.4%) novel variants, of which 2.8% were coding-synonymous, 93.3% were non-coding (64.6% intronic), and 3.8% were missense. We identified six rare IFNGR1 missense including three damaging variants (Val14Met (V14M), Val61Ile and Tyr397Cys (Y397C)) conferring a higher risk for ADEH+ (P=0.031). Variants V14M and Y397C were confirmed to be deleterious leading to partial IFNGR1 deficiency. Seven common IFNGR1 SNPs, along with common protective haplotypes (2 to 7-SNPs) conferred a reduced risk of ADEH+ (P=0.015-0.002, P=0.0015-0.0004, respectively), and both SNP and haplotype associations were replicated in an independent AA sample (P=0.004-0.0001 and P=0.001-0.0001, respectively).
Conclusion
Our results provide evidence that both genetic variants in the gene encoding IFNGR1 are implicated in susceptibility to the ADEH+ phenotype.
CAPSULE SUMMARY
We provided the first evidence that rare functional IFNGR1 mutations contribute to a defective systemic IFN-γ immune response that accounts for the propensity of AD patients to disseminated viral skin infections.
doi:10.1016/j.jaci.2015.06.047
PMCID: PMC4679503  PMID: 26343451
IFNGR1; genetic variants; atopic dermatitis; eczema herpeticum
2.  Filaggrin Mutations That Confer Risk of Atopic Dermatitis Confer Greater Risk for Eczema Herpeticum 
Background
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. Eczema herpeticum (ADEH) is a rare but serious complication of AD resulting from disseminated cutaneous HSV infections.
Objective
We aimed to determine whether FLG polymorphisms contribute to ADEH susceptibility.
Methods
Two common loss-of-function mutations plus nine FLG single nucleotide polymorphisms (SNPs) were genotyped in 278 European American AD patients, of whom 112 had ADEH, and 157non-atopic controls. Replication was performed on 339 African Americans.
Results
Significant associations were observed for both the R501X and 2282del4 mutations and AD among European Americans (P=1.46×10−5,3.87×10−5, respectively), but the frequency of the R501X mutation was three times higher (25.% vs 9%) for ADEH compared to AD without EH (odds ratio [OR]=3.4 (1.7–6.8), P=0.0002). Associations with ADEH were stronger with the combined null mutations (OR=10.1 (4.7–22.1), P=1.99×10−11). Associations with the R501X mutation were replicated in the African American population; the null mutation was absent among healthy African Americans, but present among AD (3.2%, P=0.035) and common among ADEH (9.4%; P=0.0049) patients. However, the 2282del4 mutation was absent among African American ADEH patients and rare (<1%) among healthy individuals.
Conclusion
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.
Clinical Implications
The Filaggrin (FLG) R501X Mutation, a major risk factor for atopic dermatitis, confers a greater risk of the severe, HSV-associated complication, eczema herpeticum in diverse ethnic groups.
Capsule Summary
Mutations in the skin barrier function protein, filaggrin, are strong predictors of atopic dermatitis. This report demonstrates an even greater association between one of these mutations (R501X) and eczema herpeticum in ethnically diverse American populations.
doi:10.1016/j.jaci.2009.07.034
PMCID: PMC5103856  PMID: 19733298
Atopic dermatitis; Eczema herpeticum; filaggrin; R501X; 2282del4; Single Nucleotide Polymorphisms
3.  Challenges and disparities in the application of personalized genomic medicine to populations with African ancestry 
Nature Communications  2016;7:12521.
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.
Personalized medicine requires accurate and ethnicity-optimized reference genome panels. Here, the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) evaluates typical variant filters and existing genome databases against newly sequenced African-ancestry populations.
doi:10.1038/ncomms12521
PMCID: PMC5062569  PMID: 27725664
4.  A continuum of admixture in the Western Hemisphere revealed by the African Diaspora genome 
Nature Communications  2016;7:12522.
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.
The Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) aims to better understand population genetics of the African diaspora. Here, it uses deeply sequenced whole-genomes to describe the impact of admixture and potential disease burden of deleterious variants.
doi:10.1038/ncomms12522
PMCID: PMC5062574  PMID: 27725671
5.  Hemizygous Deletion on Chromosome 3p26.1 Is Associated with Heavy Smoking among African American Subjects in the COPDGene Study 
PLoS ONE  2016;11(10):e0164134.
Many well-powered genome-wide association studies have identified genetic determinants of self-reported smoking behaviors and measures of nicotine dependence, but most have not considered the role of structural variants, such as copy number variation (CNVs), influencing these phenotypes. Here, we included 2,889 African American and 6,187 non-Hispanic White subjects from the COPDGene cohort (http://www.copdgene.org) to carefully investigate the role of polymorphic CNVs across the genome on various measures of smoking behavior. We identified a CNV component (a hemizygous deletion) on chromosome 3p26.1 associated with two quantitative phenotypes related to smoking behavior among African Americans. This polymorphic hemizygous deletion is significantly associated with pack-years and cigarettes smoked per day among African American subjects in the COPDGene study. We sought evidence of replication in African Americans from the population based Atherosclerosis Risk in Communities (ARIC) study. While we observed similar CNV counts, the extent of exposure to cigarette smoking among ARIC subjects was quite different and the smaller sample size of heavy smokers in ARIC severely limited statistical power, so we were unable to replicate our findings from the COPDGene cohort. But meta-analyses of COPDGene and ARIC study subjects strengthened our association signal. However, a few linkage studies have reported suggestive linkage to the 3p26.1 region, and a few genome-wide association studies (GWAS) have reported markers in the gene (GRM7) nearest to this 3p26.1 area of polymorphic deletions are associated with measures of nicotine dependence among subjects of European ancestry.
doi:10.1371/journal.pone.0164134
PMCID: PMC5053531  PMID: 27711239
6.  A Genome-Wide Association Study of Emphysema and Airway Quantitative Imaging Phenotypes 
Rationale: Chronic obstructive pulmonary disease (COPD) is defined by the presence of airflow limitation on spirometry, yet subjects with COPD can have marked differences in computed tomography imaging. These differences may be driven by genetic factors. We hypothesized that a genome-wide association study (GWAS) of quantitative imaging would identify loci not previously identified in analyses of COPD or spirometry. In addition, we sought to determine whether previously described genome-wide significant COPD and spirometric loci were associated with emphysema or airway phenotypes.
Objectives: To identify genetic determinants of quantitative imaging phenotypes.
Methods: We performed a GWAS on two quantitative emphysema and two quantitative airway imaging phenotypes in the COPDGene (non-Hispanic white and African American), ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints), NETT (National Emphysema Treatment Trial), and GenKOLS (Genetics of COPD, Norway) studies and on percentage gas trapping in COPDGene. We also examined specific loci reported as genome-wide significant for spirometric phenotypes related to airflow limitation or COPD.
Measurements and Main Results: The total sample size across all cohorts was 12,031, of whom 9,338 were from COPDGene. We identified five loci associated with emphysema-related phenotypes, one with airway-related phenotypes, and two with gas trapping. These loci included previously reported associations, including the HHIP, 15q25, and AGER loci, as well as novel associations near SERPINA10 and DLC1. All previously reported COPD and a significant number of spirometric GWAS loci were at least nominally (P < 0.05) associated with either emphysema or airway phenotypes.
Conclusions: Genome-wide analysis may identify novel risk factors for quantitative imaging characteristics in COPD and also identify imaging features associated with previously identified lung function loci.
doi:10.1164/rccm.201501-0148OC
PMCID: PMC4595690  PMID: 26030696
emphysema; airway; genetics; chronic obstructive pulmonary disease
7.  A Genome-wide analysis of the response to inhaled beta2-agonists in Chronic Obstructive Pulmonary Disease 
The pharmacogenomics journal  2015;16(4):326-335.
Short-acting β2-agonist bronchodilators are the most common medications used in treating chronic obstructive pulmonary disease (COPD). Genetic variants determining bronchodilator responsiveness (BDR) in COPD have not been identified.
We performed a genome-wide association study (GWAS) of BDR in 5789 current or former smokers with COPD in one African American and four white populations. BDR was defined as the quantitative spirometric response to inhaled β2-agonists. We combined results in a meta-analysis.
In the meta-analysis, SNPs in the genes KCNK1 (P=2.02×10−7) and KCNJ2 (P=1.79×10−7) were the top associations with BDR. Among African Americans, SNPs in CDH13 were significantly associated with BDR (P=5.1×10−9). A nominal association with CDH13 was identified in a gene-based analysis in all subjects.
We identified suggestive association with BDR among COPD subjects for variants near two potassium channel genes (KCNK1 and KCNJ2). SNPs in CDH13 were significantly associated with BDR in African Americans.
doi:10.1038/tpj.2015.65
PMCID: PMC4848212  PMID: 26503814
8.  Genome-Wide Association Study Identification of Novel Loci Associated with Airway Responsiveness in Chronic Obstructive Pulmonary Disease 
Increased airway responsiveness is linked to lung function decline and mortality in subjects with chronic obstructive pulmonary disease (COPD); however, the genetic contribution to airway responsiveness remains largely unknown. A genome-wide association study (GWAS) was performed using the Illumina (San Diego, CA) Human660W-Quad BeadChip on European Americans with COPD from the Lung Health Study. Linear regression models with correlated meta-analyses, including data from baseline (n = 2,814) and Year 5 (n = 2,657), were used to test for common genetic variants associated with airway responsiveness. Genotypic imputation was performed using reference 1000 Genomes Project data. Expression quantitative trait loci (eQTL) analyses in lung tissues were assessed for the top 10 markers identified, and immunohistochemistry assays assessed protein staining for SGCD and MYH15. Four genes were identified within the top 10 associations with airway responsiveness. Markers on chromosome 9p21.2 flanked by LINGO2 met a predetermined threshold of genome-wide significance (P < 9.57 × 10−8). Markers on chromosomes 3q13.1 (flanked by MYH15), 5q33 (SGCD), and 6q21 (PDSS2) yielded suggestive evidence of association (9.57 × 10−8 < P ≤ 4.6 × 10−6). Gene expression studies in lung tissue showed single nucleotide polymorphisms on chromosomes 5 and 3 to act as eQTL for SGCD (P = 2.57 × 10−9) and MYH15 (P = 1.62 × 10−6), respectively. Immunohistochemistry confirmed localization of SGCD protein to airway smooth muscle and vessels and MYH15 to airway epithelium, vascular endothelium, and inflammatory cells. We identified novel loci associated with airway responsiveness in a GWAS among smokers with COPD. Risk alleles on chromosomes 5 and 3 acted as eQTLs for SGCD and MYH15 messenger RNA, and these proteins were expressed in lung cells relevant to the development of airway responsiveness.
doi:10.1165/rcmb.2014-0198OC
PMCID: PMC4566043  PMID: 25514360
COPD; airway reactivity; bronchial responsiveness; eQTL; δ-sarcoglycan
9.  IRF6 mutation screening in nonsyndromic orofacial clefting: analysis of 1521 families 
Clinical genetics  2015;90(1):28-34.
Van der Woude syndrome (VWS) is an autosomal dominant malformation syndrome characterized by orofacial clefting (OFC) and lower lip pits. The clinical presentation of VWS is variable and can present as an isolated OFC, making it difficult to distinguish VWS cases from individuals with nonsyndromic OFCs. About 70% of causal VWS mutations occur in IRF6, a gene that is also associated with nonsyndromic OFCs. Screening for IRF6 mutations in apparently nonsyndromic cases has been performed in several modestly sized cohorts with mixed results. In the current study we screened 1521 trios with presumed nonsyndromic OFCs to determine the frequency of causal IRF6 mutations. We identified seven likely causal IRF6 mutations, although a posteriori review identified two misdiagnosed VWS families based on the presence of lip pits. We found no evidence for association between rare IRF6 polymorphisms and nonsyndromic OFCs. We combined our results with other similar studies (totaling 2,472 families) and conclude that causal IRF6 mutations are found in 0.24%-0.44% of apparently nonsyndromic OFC families. We suggest that clinical mutation screening for IRF6 be considered for certain family patterns such as families with mixed types of OFCs and/or autosomal dominant transmission.
doi:10.1111/cge.12675
PMCID: PMC4783275  PMID: 26346622
nonsyndromic oral clefts; syndromic cleft; interferon regulatory factor 6; mutation screening
10.  Genome-wide site-specific differential methylation in the blood of individuals with Klinefelter Syndrome 
Klinefelter syndrome (KS) (47 XXY) is a common sex-chromosome aneuploidy with an estimated prevalence of 1 in every 660 male births. Investigations into the associations between DNA methylation and the highly variable clinical manifestations of KS have largely focused on the supernumerary X chromosome; systematic investigations of the epigenome have been limited. We obtained genome-wide DNA methylation data from peripheral blood using the Illumina HumanMethylation450K platform in 5 KS (47 XXY), 102 male (46 XY), and 113 female (46 XX) control subjects participating in the chronic obstructive pulmonary disease (COPD) Gene Study. Empirical Bayes-mediated models were used to test for differential methylation by KS status. CpG sites with a false-discovery rate <0.05 from the first-generation HumanMethylation27K platform were further examined in an independent replication cohort of 2 KS subjects, 590 male, and 495 female controls drawn from the International COPD Genetics Network (ICGN). Differential methylation at sites throughout the genome were identified, including 86 CpG sites that were differentially methylated in KS subjects relative to both male and female controls. CpG sites annotated to the HEN1 methyltransferase homolog 1 (HENMT1), calcyclin-binding protein (CACYBP), and GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) genes were among the “KS-specific” loci that were replicated in ICGN. We therefore conclude that site-specific differential methylation exists throughout the genome in KS. The functional impact and clinical relevance of these differentially methylated loci should be explored in future studies.
doi:10.1002/mrd.22483
PMCID: PMC4439255  PMID: 25988574
[MeSH]: Klinefelter syndrome; DNA methylation; epigenomics; XXY syndrome
11.  A genome-wide study of inherited deletions identified two regions associated with non-syndromic isolated oral clefts 
Background
DNA copy number variants play an important part in the development of common birth defects such as oral clefts. Individual patients with multiple birth defects (including oral clefts) have been shown to carry small and large chromosomal deletions.
Methods
We investigated the role of polymorphic copy number deletions by comparing transmission rates of deletions from parents to offspring in case-parent trios of European ancestry ascertained through a cleft proband with trios ascertained through a normal offspring. DNA copy numbers in trios were called using the joint hidden Markov model in the freely available PennCNV software (www.openbioinformatics.org/penncnv). All statistical analyses were performed using Bioconductor tools (www.bioconductor.org) in the open source environment R.
Results
We identified a 67 kilo-base (kb) region in the gene MGAM on chromosome 7q34, and a 206 kb region overlapping genes ADAM3A and ADAM5 on chromosome 8p11, where deletions are more frequently transmitted to cleft offspring than control offspring.
Conclusions
These genes or nearby regulatory elements may be involved in the etiology of oral clefts.
doi:10.1002/bdra.23362
PMCID: PMC4415613  PMID: 25776870
Oral clefts; DNA copy numbers; inherited deletions; case-parent trios; arrays
12.  The PhenX Toolkit: Get the Most From Your Measures 
American Journal of Epidemiology  2011;174(3):253-260.
The potential for genome-wide association studies to relate phenotypes to specific genetic variation is greatly increased when data can be combined or compared across multiple studies. To facilitate replication and validation across studies, RTI International (Research Triangle Park, North Carolina) and the National Human Genome Research Institute (Bethesda, Maryland) are collaborating on the consensus measures for Phenotypes and eXposures (PhenX) project. The goal of PhenX is to identify 15 high-priority, well-established, and broadly applicable measures for each of 21 research domains. PhenX measures are selected by working groups of domain experts using a consensus process that includes input from the scientific community. The selected measures are then made freely available to the scientific community via the PhenX Toolkit. Thus, the PhenX Toolkit provides the research community with a core set of high-quality, well-established, low-burden measures intended for use in large-scale genomic studies. PhenX measures will have the most impact when included at the experimental design stage. The PhenX Toolkit also includes links to standards and resources in an effort to facilitate data harmonization to legacy data. Broad acceptance and use of PhenX measures will promote cross-study comparisons to increase statistical power for identifying and replicating variants associated with complex diseases and with gene-gene and gene-environment interactions.
doi:10.1093/aje/kwr193
PMCID: PMC3141081  PMID: 21749974
environmental exposure; epidemiologic methods; genetic research; genetics; genome-wide association study; meta-analysis as topic; phenotype; research design
13.  Clinical and Radiologic Disease in Smokers With Normal Spirometry 
JAMA internal medicine  2015;175(9):1539-1549.
IMPORTANCE
Airflow obstruction on spirometry is universally used to define chronic obstructive pulmonary disease (COPD), and current or former smokers without airflow obstruction may assume that they are disease free.
OBJECTIVE
To identify clinical and radiologic evidence of smoking-related disease in a cohort of current and former smokers who did not meet spirometric criteria for COPD, for whom we adopted the discarded label of Global Initiative for Obstructive Lung Disease (GOLD) 0.
DESIGN, SETTING, AND PARTICIPANTS
Individuals from the Genetic Epidemiology of COPD (COPDGene) cross-sectional observational study completed spirometry, chest computed tomography (CT) scans, a 6-minute walk, and questionnaires. Participants were recruited from local communities at 21 sites across the United States. The GOLD 0 group (n = 4388) (ratio of forced expiratory volume in the first second of expiration [FEV1] to forced vital capacity >0.7 and FEV1 ≥80% predicted) from the COPDGene study was compared with a GOLD 1 group (n = 794), COPD groups (n = 3690), and a group of never smokers (n = 108). Recruitment began in January 2008 and ended in July 2011.
MAIN OUTCOMES AND MEASURES
Physical function impairments, respiratory symptoms, CT abnormalities, use of respiratory medications, and reduced respiratory-specific quality of life.
RESULTS
One or more respiratory-related impairments were found in 54.1% (2375 of 4388) of the GOLD 0 group. The GOLD 0 group had worse quality of life (mean [SD] St George’s Respiratory Questionnaire total score, 17.0 [18.0] vs 3.8 [6.8] for the never smokers; P < .001) and a lower 6-minute walk distance, and 42.3% (127 of 300) of the GOLD 0 group had CT evidence of emphysema or airway thickening. The FEV1 percent predicted distribution and mean for the GOLD 0 group were lower but still within the normal range for the population. Current smoking was associated with more respiratory symptoms, but former smokers had greater emphysema and gas trapping. Advancing age was associated with smoking cessation and with more CT findings of disease. Individuals with respiratory impairments were more likely to use respiratory medications, and the use of these medications was associated with worse disease.
CONCLUSIONS AND RELEVANCE
Lung disease and impairments were common in smokers without spirometric COPD. Based on these results, we project that there are 35 million current and former smokers older than 55 years in the United States who may have unrecognized disease or impairment. The effect of chronic smoking on the lungs and the individual is substantially underestimated when using spirometry alone.
doi:10.1001/jamainternmed.2015.2735
PMCID: PMC4564354  PMID: 26098755
14.  Genetic control of gene expression at novel and established chronic obstructive pulmonary disease loci 
Human Molecular Genetics  2014;24(4):1200-1210.
Genetic risk loci have been identified for a wide range of diseases through genome-wide association studies (GWAS), but the relevant functional mechanisms have been identified for only a small proportion of these GWAS-identified loci. By integrating results from the largest current GWAS of chronic obstructive disease (COPD) with expression quantitative trait locus (eQTL) analysis in whole blood and sputum from 121 subjects with COPD from the ECLIPSE Study, this analysis identifies loci that are simultaneously associated with COPD and the expression of nearby genes (COPD eQTLs). After integrative analysis, 19 COPD eQTLs were identified, including all four previously identified genome-wide significant loci near HHIP, FAM13A, and the 15q25 and 19q13 loci. For each COPD eQTL, fine mapping and colocalization analysis to identify causal shared eQTL and GWAS variants identified a subset of sites with moderate-to-strong evidence of harboring at least one shared variant responsible for both the eQTL and GWAS signals. Transcription factor binding site (TFBS) analysis confirms that multiple COPD eQTL lead SNPs disrupt TFBS, and enhancer enrichment analysis for loci with the strongest colocalization signals showed enrichment for blood-related cell types (CD3 and CD4+ T cells, lymphoblastoid cell lines). In summary, integrative eQTL and GWAS analysis confirms that genetic control of gene expression plays a key role in the genetic architecture of COPD and identifies specific blood-related cell types as likely participants in the functional pathway from GWAS-associated variant to disease phenotype.
doi:10.1093/hmg/ddu525
PMCID: PMC4806382  PMID: 25315895
15.  Dissecting genetics for chronic mucus hypersecretion in smokers with and without COPD 
Background
Smoking is a notorious risk factor for chronic mucus hypersecretion (CMH). CMH frequently occurs in Chronic Obstructive Pulmonary Disease (COPD). The question arises whether the same single nucleotide polymorphisms (SNPs) are related to CMH in smokers with and without COPD.
Methods
We performed two genome wide association studies on CMH under an additive genetic model in male heavy smokers (≥20 pack-years) with COPD (n=849, 39.9% CMH) and without COPD (n=1,348, 25.4% CMH), followed by replication and meta-analysis in comparable populations, and assessment of the functional relevance of significantly associated SNPs.
Results
GWA analysis on CMH in COPD and non-COPD yielded no genome wide significance after replication. In COPD, our top SNP (rs10461985, p=5.43×10−5) was located in the GDNF-antisense gene that is functionally associated with the GDNF gene. Expression of GDNF in bronchial biopsies of COPD patients was significantly associated with CMH (p=0.007). In non-COPD, 4 SNPs had a p-value <10−5 in the meta-analysis, including a SNP (rs4863687) in the MAML3 gene, the T-allele showing modest association with CMH (p=7.57×10−6, OR=1.48) and with significantly increased MAML3 expression in lung tissue (p=2.59×10−12).
Conclusions
Our data suggest the potential for differential genetic backgrounds of CMH in individuals with and without COPD.
doi:10.1183/09031936.00093314
PMCID: PMC4498483  PMID: 25234806
16.  X- linked markers in DMD associated with oral clefts 
As part of an international consortium, case-parent trios were collected for a genome wide association study of isolated, non-syndromic oral clefts, including cleft lip (CL), cleft palate (CP) and cleft lip and palate (CLP). Non-syndromic oral clefts have a complex and heterogeneous etiology. Risk is influenced by genes, environmental factors, and differs markedly by gender. Family based association tests (FBAT) were used on 14,486 SNPs spanning the X chromosome, stratified by type of cleft and racial group. Significant results even after multiple comparisons correction were obtained for the Duchene’s muscular dystrophy (DMD) gene, the largest single gene in the human genome, among CL/P trios (both CL and CLP combined). When stratified into groups of European and Asian ancestry, stronger signals were obtained for Asians. Although conventional sliding window haplotype analysis showed no increase in significance, analysis selected combinations of the 25 most significant SNPs in DMD identified four SNPs together that attained genome-wide significance among Asian CL/P trios, raising the possibility of interaction between distant SNPs within DMD.
doi:10.1111/eos.12025
PMCID: PMC3600648  PMID: 23489894
oral clefts; case-parent trios; X-linked; family-based association; DMD
17.  A genome-wide association study identifies risk loci for spirometric measures among smokers of European and African ancestry 
BMC Genetics  2015;16:138.
Background
Pulmonary function decline is a major contributor to morbidity and mortality among smokers. Post bronchodilator FEV1 and FEV1/FVC ratio are considered the standard assessment of airflow obstruction. We performed a genome-wide association study (GWAS) in 9919 current and former smokers in the COPDGene study (6659 non-Hispanic Whites [NHW] and 3260 African Americans [AA]) to identify associations with spirometric measures (post-bronchodilator FEV1 and FEV1/FVC). We also conducted meta-analysis of FEV1 and FEV1/FVC GWAS in the COPDGene, ECLIPSE, and GenKOLS cohorts (total n = 13,532).
Results
Among NHW in the COPDGene cohort, both measures of pulmonary function were significantly associated with SNPs at the 15q25 locus [containing CHRNA3/5, AGPHD1, IREB2, CHRNB4] (lowest p-value = 2.17 × 10−11), and FEV1/FVC was associated with a genomic region on chromosome 4 [upstream of HHIP] (lowest p-value = 5.94 × 10−10); both regions have been previously associated with COPD. For the meta-analysis, in addition to confirming associations to the regions near CHRNA3/5 and HHIP, genome-wide significant associations were identified for FEV1 on chromosome 1 [TGFB2] (p-value = 8.99 × 10−9), 9 [DBH] (p-value = 9.69 × 10−9) and 19 [CYP2A6/7] (p-value = 3.49 × 10−8) and for FEV1/FVC on chromosome 1 [TGFB2] (p-value = 8.99 × 10−9), 4 [FAM13A] (p-value = 3.88 × 10−12), 11 [MMP3/12] (p-value = 3.29 × 10−10) and 14 [RIN3] (p-value = 5.64 × 10−9).
Conclusions
In a large genome-wide association study of lung function in smokers, we found genome-wide significant associations at several previously described loci with lung function or COPD. We additionally identified a novel genome-wide significant locus with FEV1 on chromosome 9 [DBH] in a meta-analysis of three study populations.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-015-0299-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12863-015-0299-4
PMCID: PMC4668640  PMID: 26634245
Chronic obstructive pulmonary disease; DBH; FEV1; FEV1/FVC; Genome-wide association study; Spirometry
18.  Analytic power and sample size calculation for the genotypic transmission/disequilibrium test in case-parent trio studies 
Case-parent trio studies considering genotype data from children affected by a disease and from their parents are frequently used to detect single nucleotide polymorphisms (SNPs) associated with disease. The most popular statistical tests in this study design are transmission/disequlibrium tests (TDTs). Several types of these tests have been developed, e.g., procedures based on alleles or genotypes. Therefore, it is of great interest to examine which of these tests have the highest statistical power to detect SNPs associated with disease. Comparisons of the allelic and the genotypic TDT for individual SNPs have so far been conducted based on simulation studies, since the test statistic of the genotypic TDT was determined numerically. Recently, it, however, has been shown that this test statistic can be presented in closed form. In this article, we employ this analytic solution to derive equations for calculating the statistical power and the required sample size for different types of the genotypic TDT. The power of this test is then compared with the one of the corresponding score test assuming the same mode of inheritance as well as the allelic TDT based on a multiplicative mode of inheritance, which is equivalent to the score test assuming an additive mode of inheritance. This is, thus, the first time that the power of these tests are compared based on equations, yielding instant results and omitting the need for time-consuming simulation studies. This comparison reveals that the tests have almost the same power, with the score test being slightly more powerful.
doi:10.1002/bimj.201300148
PMCID: PMC4206700  PMID: 25123830
Case-parent trio design; Conditional logisitc regression; Genome-wide association studies; Power calculation; Wald test
19.  Common Genetic Variants Associated with Resting Oxygenation in Chronic Obstructive Pulmonary Disease 
Hypoxemia is a major complication of chronic obstructive pulmonary disease (COPD) that correlates with disease prognosis. Identifying genetic variants associated with oxygenation may provide clues for deciphering the heterogeneity in prognosis among patients with COPD. However, previous genetic studies have been restricted to investigating COPD candidate genes for association with hypoxemia. To report results from the first genome-wide association study (GWAS) of resting oxygen saturation (as measured by pulse oximetry [Spo2]) in subjects with COPD, we performed a GWAS of Spo2 in two large, well characterized COPD populations: COPDGene, including both the non-Hispanic white (NHW) and African American (AA) groups, and Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE). We identified several suggestive loci (P < 1 × 10−5) associated with Spo2 in COPDGene in the NHW (n = 2810) and ECLIPSE (n = 1758) groups, and two loci on chromosomes 14 and 15 in the AA group (n = 820) from COPDGene achieving a level of genome-wide significance (P < 5 × 10−8). The chromosome 14 single-nucleotide polymorphism, rs6576132, located in an intergenic region, was nominally replicated (P < 0.05) in the NHW group from COPDGene. The chromosome 15 single-nucleotide polymorphisms were rare in subjects of European ancestry, so the results could not be replicated. The chromosome 15 region contains several genes, including TICRR and KIF7, and is proximal to RHCG (Rh family C glyocoprotein gene). We have identified two loci associated with resting oxygen saturation in AA subjects with COPD, and several suggestive regions in subjects of European descent with COPD. Our study highlights the importance of investigating the genetics of complex traits in different racial groups.
doi:10.1165/rcmb.2014-0135OC
PMCID: PMC4224086  PMID: 24825563
chronic obstructive pulmonary disease; hypoxemia; pulse oximetry; genome-wide association study; oxygen saturation
20.  Oesophageal squamous cell carcinoma in high-risk Chinese populations: Possible role for vascular epithelial growth factor A 
Background
Mechanisms involved in wound healing play some role in carcinogenesis in multiple organs, likely by creating a chronic inflammatory milieu. This study sought to assess the role of genetic markers in selected inflammation-related genes involved in wound healing (interleukin (IL)-1a, IL-1b, IL-1 Receptor type I (IL-1Ra), IL-1 Receptor type II (IL-1Rb), tumour necrosis factor (TNF)-α, tumour necrosis factor receptor superfamily member (TNFRSF)1A, nuclear factor kappa beta (NF-kB)1, NF-kB2, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, hypoxia induced factor (HIF)-1α, vascular endothelial growth factor (VEGF)A and P-53) in risk to oesophageal squamous cell carcinoma (OSCC).
Methods
We genotyped 125 tag single nucleotide polymorphism (SNP)s in 410 cases and 377 age and sex matched disease-free individuals from Nutritional Intervention Trial (NIT) cohort, and 546 cases and 556 controls individually matched for age, sex and neighbourhood from Shanxi case–control study, both conducted in high-risk areas of north-central China (1985–2007). Cox proportional-hazard models and conditional logistic regression models were used for SNPs analyses for NIT and Shanxi, respectively. Fisher's inverse test statistics were used to obtain gene-level significance.
Results
Multiple SNPs were significantly associated with OSCC in both studies, however, none retained their significance after a conservative Bonferroni adjustment. Empiric p-values for tag SNPs in VEGFA in NIT were highly concentrated in the lower tail of the distribution, suggesting this gene may be influencing risk. Permutation tests confirmed the significance of this pattern. At the gene level, VEGFA yielded an empiric significance (P = 0.027) in NIT. We also observed some evidence for interaction between environmental factors and some VEGFA tag SNPs.
Conclusion
Our finding adds further evidence for a potential role for markers in the VEGFA gene in the development and progression of early precancerous lesions of oesophagus.
doi:10.1016/j.ejca.2014.07.022
PMCID: PMC4363989  PMID: 25172294
Oesophageal squamous; cell carcinoma; Inflammation; Wound-healing; Genetic marker; Genetics; Inflammation-related events; Vascular endothelial growth factor A; VEGFA
21.  Admixture Mapping Identifies a Quantitative Trait Locus Associated with FEV1/FVC in the COPDGene Study 
Genetic epidemiology  2014;38(7):652-659.
African Americans are admixed with genetic contributions from European and African ancestral populations. Admixture mapping leverages this information to map genes influencing differential disease risk across populations. We performed admixture and association mapping in 3300 African American current or former smokers from the COPDGene Study. We analyzed estimated local ancestry and SNP genotype information to identify regions associated with FEV1/FVC, the ratio of forced expiratory volume in one second to forced vital capacity, measured by spirometry performed after bronchodilator administration. Global African ancestry inversely associated with FEV1/FVC (p = 0.035). Genome-wide admixture analysis, controlling for age, gender, body mass index, current smoking status, pack-years smoked, and four principal components summarizing the genetic background of African Americans in the COPDGene Study, identified a region on chromosome 12q14.1 associated with FEV1/FVC (p = 2.1 × 10-6) when regressed on local ancestry. Allelic association in this region of chromosome 12 identified an intronic variant in FAM19A2 (rs348644) as associated with FEV1/FVC (p=1.76 × 10-6). By combining admixture and association mapping, a marker on chromosome 12q14.1 was identified as being associated with reduced FEV1/FVC ratio among African-Americans in the COPDGene Study.
doi:10.1002/gepi.21847
PMCID: PMC4190160  PMID: 25112515
admixture mapping; lung function; COPD; African Americans
22.  Genome-wide interaction studies reveal sex-specific asthma risk alleles 
Human Molecular Genetics  2014;23(19):5251-5259.
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.
doi:10.1093/hmg/ddu222
PMCID: PMC4159149  PMID: 24824216
23.  Dectecting disease variants in case-parent trio studies using the Bioconductor software package trio 
Genetic epidemiology  2014;38(6):516-522.
Case-parent trio studies are commonly employed in genetics to detect variants underlying common complex disease risk. Both commercial and freely available software suites for genetic data analysis usually contain methods for case-parent trio designs. A user might, however, experience limitations with these packages, which can include missing functionality to extend the software if a desired analysis has not been implemented, and the inability to programmatically capture all the software versions used for low-level processing and high-level inference of genomic data, a critical consideration in particular for high-throughput experiments. Here, we present a software vignette (i.e., a manual with step by step instructions and examples to demonstrate software functionality) for reproducible genome-wide analyses of case-parent trio data using the open source Bioconductor package trio. The workflow for the practitioner uses data from previous genetic trio studies to illustrate functions for marginal association tests, assessment of parent-of-origin effects, power and sample size calculations, and functions to detect gene-gene and gene-environment interactions associated with disease.
doi:10.1002/gepi.21836
PMCID: PMC4139708  PMID: 25048299
Software; Case-parent trios; Transmission disequilibrium tests; Gene-environment interactions; Parent-of-origin effects
24.  Beyond GWAS in COPD: Probing the landscape between gene-set associations, genome-wide associations and protein-protein interaction networks 
Human heredity  2014;78(0):131-139.
Objectives
To use a systems biology approach to integrate genotype and protein-protein interaction (PPI) data to identify disease network modules associated with chronic obstructive pulmonary disease (COPD) and to perform traditional pathway analysis.
Methods
We used a standard gene-set association approach (FORGE) using gene-based association analysis and gene-set definitions from the molecular signatures database (MSigDB). As a discovery step we analyzed GWAS results from two well-characterized COPD cohorts, COPDGene and GenKOLS. We used a third well-characterized COPD case-control cohort for replication, ECLIPSE. Next, we used dmGWAS, a method that integrates GWAS results with PPI, to identify COPD disease modules.
Results
No gene-sets reached experiment-wide significance in either discovery population. We identified a consensus network of 10 genes identified in modules by integrating GWAS results with PPI that replicated in COPDGene, GenKOLS, and ECLIPSE. Members of four gene-sets were enriched among these 10 genes: (i) lung adenocarcinoma tumor sequencing genes, (ii) IL7 pathway genes, (iii) kidney cell response to arsenic, and (iv) CD4 T cell responses. Further, several genes have also been associated with pathophysiology relevant to COPD including KCNK3, NEDD4L and RIN3. In particular, KCNK3 has been associated with pulmonary arterial hypertension, a common complication in advanced COPD.
Conclusion
We report a set of new genes that may influence the etiology of COPD that would not have been identified using traditional GWAS and pathway analyses alone.
doi:10.1159/000365589
PMCID: PMC4415367  PMID: 25171373
COPD; genome-wide scan; protein-protein interaction network; gene-set association; pathway association; disease module
25.  Genome-wide Association Study Identifies Peanut Allergy-Specific Loci and Evidence of Epigenetic Mediation in U.S. Children 
Nature communications  2015;6:6304.
Food allergy (FA) affects 2–10% of U.S. children and is a growing clinical and public health problem. Here we conduct the first genome-wide association study of well-defined FA, including specific subtypes (peanut, milk, and egg) in 2,759 U.S. participants (1,315 children; 1,444 parents) from the Chicago Food Allergy Study; and identify peanut allergy (PA)-specific loci in the HLA-DR and -DQ gene region at 6p21.32, tagged by rs7192 (p=5.5×10−8) and rs9275596 (p=6.8×10−10), in 2,197 participants of European ancestry. We replicate these associations in an independent sample of European ancestry. These associations are further supported by meta-analyses across the discovery and replication samples. Both single-nucleotide polymorphisms (SNPs) are associated with differential DNA methylation levels at multiple CpG sites (p<5×10−8); and differential DNA methylation of the HLA-DQB1 and HLA-DRB1 genes partially mediate the identified SNP-PA associations. This study suggests that the HLA-DR and -DQ gene region likely poses significant genetic risk for PA.
doi:10.1038/ncomms7304
PMCID: PMC4340086  PMID: 25710614

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