Ivana Nedeljkovic

Genome-wide association study on the FEV1/FVC ratio in never-smokers identifies HHIP and FAM13A.

Background: Although a striking proportion (25% to 45%) of patients with chronic obstructive pulmonary disease are never‐smokers, most genetic susceptibility studies have not focused on this group exclusively. Objective: The aim of this study was to identify common genetic variants associated with FEV1 and its ratio to forced vital capacity (FVC) in never‐smokers. Methods: Genome‐wide association studies were performed in 5070 never‐smokers of the identification cohort LifeLines, and results (P < 10−5) were verified by using a meta‐analysis of the Vlagtwedde‐Vlaardingen study and the Rotterdam Study I‐III (total n = 1966). Furthermore, we aimed to assess the effects of the replicated variants in more detail by performing genetic risk score, expression quantitative trait loci, and variant*ever‐smoking interaction analyses. Results: We identified associations between the FEV1/FVC ratio and 5 common genetic variants in the identification cohort, and 2 of these associations were replicated. The 2 variants annotated to the genes hedgehog interacting protein (HHIP) and family with sequence similarity 13 member A (FAM13A) were shown to have an additive effect on FEV1/FVC levels in the genetic risk score analysis; were associated with gene expression of HHIP and FAM13A in lung tissue, respectively; and were genome‐wide significant in a meta‐analysis including both identification and 4 verification cohorts (P < 2.19 × 10−7). Finally, we did not identify significant interactions between the variants and ever smoking. Results of the FEV1 identification analysis were not replicated. Conclusion: The genes HHIP and FAM13A confer a risk for airway obstruction in general that is not driven exclusively by cigarette smoking, which is the main risk factor for chronic obstructive pulmonary disease.

Long-term Air Pollution Exposure, Genome-wide DNA Methylation and Lung Function in the LifeLines Cohort Study

Background: Long-term air pollution exposure is negatively associated with lung function, yet the mechanisms underlying this association are not fully clear. Differential DNA methylation may explain this association. Objectives: Our main aim was to study the association between long-term air pollution exposure and DNA methylation. Methods: We performed a genome-wide methylation study using robust linear regression models in 1,017 subjects from the LifeLines cohort study to analyze the association between exposure to nitrogen dioxide (NO2) and particulate matter (PM2.5, fine particulate matter with aerodynamic diameter ≤2.5μm; PM10, particulate matter with aerodynamic diameter ≤10μm) and PM2.5absorbance, indicator of elemental carbon content (estimated with land-use-regression models) with DNA methylation in whole blood (Illumina® HumanMethylation450K BeadChip). Replication of the top hits was attempted in two independent samples from the population-based Cooperative Health Research in the Region of Augsburg studies (KORA). Results: Depending on the p-value threshold used, we found significant associations between NO2 exposure and DNA methylation for seven CpG sites (Bonferroni corrected threshold p<1.19×10−7) or for 4,980 CpG sites (False Discovery Rate<0.05). The top associated CpG site was annotated to the PSMB9 gene (i.e., cg04908668). None of the seven Bonferroni significant CpG-sites were significantly replicated in the two KORA-cohorts. No associations were found for PM exposure. Conclusions: Long-term NO2 exposure was genome-wide significantly associated with DNA methylation in the identification cohort but not in the replication cohort. Future studies are needed to further elucidate the potential mechanisms underlying NO2-exposure–related respiratory disease. https://doi.org/10.1289/EHP2045

Occupational exposure to pesticides is associated with differential DNA methylation

Objectives Occupational pesticide exposure is associated with a wide range of diseases, including lung diseases, but it is largely unknown how pesticides influence airway disease pathogenesis. A potential mechanism might be through epigenetic mechanisms, like DNA methylation. Therefore, we assessed associations between occupational exposure to pesticides and genome-wide DNA methylation sites. Methods 1561 subjects of LifeLines were included with either no (n=1392), low (n=108) or high (n=61) exposure to any type of pesticides (estimated based on current or last held job). Blood DNA methylation levels were measured using Illumina 450K arrays. Associations between pesticide exposure and 420 938 methylation sites (CpGs) were assessed using robust linear regression adjusted for appropriate confounders. In addition, we performed genome-wide stratified and interaction analyses by gender, smoking and airway obstruction status, and assessed associations between gene expression and methylation for genome-wide significant CpGs (n=2802). Results In total for all analyses, high pesticide exposure was genome-wide significantly (false discovery rate P<0.05) associated with differential DNA methylation of 31 CpGs annotated to 29 genes. Twenty of these CpGs were found in subjects with airway obstruction. Several of the identified genes, for example, RYR1, ALLC, PTPRN2, LRRC3B, PAX2 and VTRNA2-1, are genes previously linked to either pesticide exposure or lung-related diseases. Seven out of 31 CpGs were associated with gene expression levels. Conclusions We show for the first time that occupational exposure to pesticides is genome-wide associated with differential DNA methylation. Further research should reveal whether this differential methylation plays a role in the airway disease pathogenesis induced by pesticides.

COPD GWAS variant at 19q13.2 in relation with DNA methylation and gene expression

Abstract Chronic obstructive pulmonary disease (COPD) is among the major health burdens in adults. While cigarette smoking is the leading risk factor, a growing number of genetic variations have been discovered to influence disease susceptibility. Epigenetic modifications may mediate the response of the genome to smoking and regulate gene expression. Chromosome 19q13.2 region is associated with both smoking and COPD, yet its functional role is unclear. Our study aimed to determine whether rs7937 (RAB4B, EGLN2), a top genetic variant in 19q13.2 region identified in genome-wide association studies of COPD, is associated with differential DNA methylation in blood (N = 1490) and gene expression in blood (N = 721) and lungs (N = 1087). We combined genetic and epigenetic data from the Rotterdam Study (RS) to perform the epigenome-wide association analysis of rs7937. Further, we used genetic and transcriptomic data from blood (RS) and from lung tissue (Lung expression quantitative trait loci mapping study), to perform the transcriptome-wide association study of rs7937. Rs7937 was significantly (FDR < 0.05) and consistently associated with differential DNA methylation in blood at 4 CpG sites in cis, independent of smoking. One methylation site (cg11298343-EGLN2) was also associated with COPD (P = 0.001). Additionally, rs7937 was associated with gene expression levels in blood in cis (EGLN2), 42% mediated through cg11298343, and in lung tissue, in cis and trans (NUMBL, EGLN2, DNMT3A, LOC101929709 and PAK2). Our results suggest that changes of DNA methylation and gene expression may be intermediate steps between genetic variants and COPD, but further causal studies in lung tissue should confirm this hypothesis.

Life-Course Genome-wide Association Study Meta-analysis of Total Body BMD and Assessment of Age-Specific Effects.

Bone mineral density (BMD) assessed by DXA is used to evaluate bone health. In children, total body (TB) measurements are commonly used; in older individuals, BMD at the lumbar spine (LS) and femoral neck (FN) is used to diagnose osteoporosis. To date, genetic variants in more than 60 loci have been identified as associated with BMD. To investigate the genetic determinants of TB-BMD variation along the life course and test for age-specific effects, we performed a meta-analysis of 30 genome-wide association studies (GWASs) of TB-BMD including 66,628 individuals overall and divided across five age strata, each spanning 15 years. We identified variants associated with TB-BMD at 80 loci, of which 36 have not been previously identified; overall, they explain approximately 10% of the TB-BMD variance when combining all age groups and influence the risk of fracture. Pathway and enrichment analysis of the association signals showed clustering within gene sets implicated in the regulation of cell growth and SMAD proteins, overexpressed in the musculoskeletal system, and enriched in enhancer and promoter regions. These findings reveal TB-BMD as a relevant trait for genetic studies of osteoporosis, enabling the identification of variants and pathways influencing different bone compartments. Only variants in ESR1 and close proximity to RANKL showed a clear effect dependency on age. This most likely indicates that the majority of genetic variants identified influence BMD early in life and that their effect can be captured throughout the life course.

DNA Methylation Signatures of Depressive Symptoms in Middle-aged and Elderly Persons: Meta-analysis of Multiethnic Epigenome-wide Studies

Importance Depressive disorders arise from a combination of genetic and environmental risk factors. Epigenetic disruption provides a plausible mechanism through which gene-environment interactions lead to depression. Large-scale, epigenome-wide studies on depression are missing, hampering the identification of potentially modifiable biomarkers. Objective To identify epigenetic mechanisms underlying depression in middle-aged and elderly persons, using DNA methylation in blood. Design, Setting, and Participants To date, the first cross-ethnic meta-analysis of epigenome-wide association studies (EWAS) within the framework of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium was conducted. The discovery EWAS included 7948 individuals of European origin from 9 population-based cohorts. Participants who were assessed for both depressive symptoms and whole-blood DNA methylation were included in the study. Results of EWAS were pooled using sample-size weighted meta-analysis. Replication of the top epigenetic sites was performed in 3308 individuals of African American and European origin from 2 population-based cohorts. Main Outcomes and Measures Whole-blood DNA methylation levels were assayed with Illumina-Infinium Human Methylation 450K BeadChip and depressive symptoms were assessed by questionnaire. Results The discovery cohorts consisted of 7948 individuals (4104 [51.6%] women) with a mean (SD) age of 65.4 (5.8) years. The replication cohort consisted of 3308 individuals (2456 [74.2%] women) with a mean (SD) age of 60.3 (6.4) years. The EWAS identified methylation of 3 CpG sites to be significantly associated with increased depressive symptoms: cg04987734 (P = 1.57 × 10−08; n = 11 256; CDC42BPB gene), cg12325605 (P = 5.24 × 10−09; n = 11 256; ARHGEF3 gene), and an intergenic CpG site cg14023999 (P = 5.99 × 10−08; n = 11 256; chromosome = 15q26.1). The predicted expression of the CDC42BPB gene in the brain (basal ganglia) (effect, 0.14; P = 2.7 × 10−03) and of ARHGEF3 in fibroblasts (effect, −0.48; P = 9.8 × 10−04) was associated with major depression. Conclusions and Relevance This study identifies 3 methylated sites associated with depressive symptoms. All 3 findings point toward axon guidance as the common disrupted pathway in depression. The findings provide new insights into the molecular mechanisms underlying the complex pathophysiology of depression. Further research is warranted to determine the utility of these findings as biomarkers of depression and evaluate any potential role in the pathophysiology of depression and their downstream clinical effects.

A Genome-Wide Linkage Study for Chronic Obstructive Pulmonary Disease in a Dutch Genetic Isolate Identifies Novel Rare Candidate Variants

Chronic obstructive pulmonary disease (COPD) is a complex and heritable disease, associated with multiple genetic variants. Specific familial types of COPD may be explained by rare variants, which have not been widely studied. We aimed to discover rare genetic variants underlying COPD through a genome-wide linkage scan. Affected-only analysis was performed using the 6K Illumina Linkage IV Panel in 142 cases clustered in 27 families from a genetic isolate, the Erasmus Rucphen Family (ERF) study. Potential causal variants were identified by searching for shared rare variants in the exome-sequence data of the affected members of the families contributing most to the linkage peak. The identified rare variants were then tested for association with COPD in a large meta-analysis of several cohorts. Significant evidence for linkage was observed on chromosomes 15q14–15q25 [logarithm of the odds (LOD) score = 5.52], 11p15.4–11q14.1 (LOD = 3.71) and 5q14.3–5q33.2 (LOD = 3.49). In the chromosome 15 peak, that harbors the known COPD locus for nicotinic receptors, and in the chromosome 5 peak we could not identify shared variants. In the chromosome 11 locus, we identified four rare (minor allele frequency (MAF) <0.02), predicted pathogenic, missense variants. These were shared among the affected family members. The identified variants localize to genes including neuroblast differentiation-associated protein (AHNAK), previously associated with blood biomarkers in COPD, phospholipase C Beta 3 (PLCB3), shown to increase airway hyper-responsiveness, solute carrier family 22-A11 (SLC22A11), involved in amino acid metabolism and ion transport, and metallothionein-like protein 5 (MTL5), involved in nicotinate and nicotinamide metabolism. Association of SLC22A11 and MTL5 variants were confirmed in the meta-analysis of 9,888 cases and 27,060 controls. In conclusion, we have identified novel rare variants in plausible genes related to COPD. Further studies utilizing large sample whole-genome sequencing should further confirm the associations at chromosome 11 and investigate the chromosome 15 and 5 linked regions.

Understanding the role of the chromosome 15q25.1 in COPD through epigenetics and transcriptomics

Chronic obstructive pulmonary disease (COPD) is a major health burden in adults and cigarette smoking is considered the most important environmental risk factor of COPD. Chromosome 15q25.1 locus is associated with both COPD and smoking. Our study aims at understanding the mechanism underlying the association of chromosome 15q25.1 with COPD through epigenetic and transcriptional variation in a population-based setting. To assess if COPD-associated variants in 15q25.1 are methylation quantitative trait loci, epigenome-wide association analysis of four genetic variants, previously associated with COPD (P < 5 × 10−8) in the 15q25.1 locus (rs12914385:C>T-CHRNA3, rs8034191:T>C-HYKK, rs13180:C>T-IREB2 and rs8042238:C>T-IREB2), was performed in the Rotterdam study (n = 1489). All four variants were significantly associated (P < 1.4 × 10−6) with blood DNA methylation of IREB2, CHRNA3 and PSMA4, of which two, including IREB2 and PSMA4, were also differentially methylated in COPD cases and controls (P < 0.04). Further additive and multiplicative effects of smoking were evaluated and no significant effect was observed. To evaluate if these four genetic variants are expression quantitative trait loci, transcriptome-wide association analysis was performed in 1087 lung samples. All four variants were also significantly associated with differential expression of the IREB2 3’UTR in lung tissues (P < 5.4 × 10−95). We conclude that regulatory mechanisms affecting the expression of IREB2 gene, such as DNA methylation, may explain the association between genetic variants in chromosome 15q25.1 and COPD, largely independent of smoking.

DNA methylation mediates the association between occupational exposures and lung function

Introduction: Occupational exposures, such as biological dust, mineral dust and gases/fumes, are associated with lower lung function levels and attribute to 15-20% of all Chronic Obstructive Pulmonary Disease (COPD) cases. Epigenetic mechanisms such as DNA methylation have been suggested to play a role in these associations. Aim: To assess if the association between occupational exposures and lung function (FEV1/FVC) is mediated by DNA methylation. Methods: We included 1,561 subjects of the LifeLines cohort with either no, low, or high occupational exposure to biological dust, mineral dust and gases/fumes based on the current or last held job. Associations between the three exposures and 420,938 blood DNA methylation sites (CpGs, Illumina 450K array) were assessed using robust linear regression adjusted for appropriate confounders. Differentially methylated regions (DMRs) were identified using comb-p in python. Mediation of the top site per region was assessed using bootstrapping in R. Results: Using p<10−5 4, 5 and 6 single CpGs were associated with biological dust, mineral dust and gases/fumes, respectively, but none were genome-wide significant. In total 7, 8, and 30 genome-wide significant DMRs were identified, respectively. The CpG cg06462684 in the promoter of YWHAH, who binds to the glucocorticoid receptor, significantly mediated the association between FEV1/FVC and mineral dust. In addition, cg14870271 in the promoter of LGALS3BP, known to play a role in immune responses, mediated the association between FEV1/FVC and gases/fumes. Conclusion: We show that the association between lung function levels and occupational exposure to mineral dust or gases/fumes is mediated by DNA methylation.

A genome-wide linkage study for COPD in a Dutch genetic isolate

Chronic obstructive pulmonary disease (COPD) is a complex and heritable disease, associated with common genetic variants. Familial types of COPD may be explained by rare variants, which have not been widely studied. We aimed to discover rare genetic variants underlying COPD through genome-wide linkage scan. Affected-only analysis was performed using 6K Illumina Linkage IV Panel in 142 cases clustered in 27 families from a genetic isolate, Erasmus Rucphen Family (ERF) study. Potential causal variants were then selected in exome sequence data from the affected members of the families that contributed the most to the peak. The shared rare variants were further tested for association with COPD in the entire ERF study (N=657). Significant evidence for linkage was observed on chromosomes 15 (log of odds (LOD) score=5.52) and 11 (LOD=3.71). Chromosome 15 is a known COPD locus harbouring nicotinic receptors. In the chromosome 11 locus we identified several rare (minor allele frequency (MAF)<0.02) predicted pathogenic variants. They were shared among the affected family members and associated with COPD in the entire ERF study (P<0.03). The identified variants localize to genes including solute carrier family (SLC22A11), involved in the amino acids metabolism and ion transport; metallothionein-like protein 5 (MTL5), involved in nicotinate and nicotinamide metabolism; neuroblast differentiation-associated protein (AHNAK), reported by GWAS of blood biomarkers in COPD and Phospholipase C Beta 3 (PLCB3), reported by GWAS of asthma. In conclusion, we have identified novel rare variants in plausible genes related to COPD, which will be further explored by replication using exome sequencing in a large independent population-based cohort.