Diana A. van der Plaat

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.

Air pollution exposure is associated with restrictive ventilatory patterns

Exposure to ambient air pollution is associated with a substantial burden of morbidity and mortality worldwide [1]. In a recent paper, Adam et al. [2] showed significantly impaired levels of forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) associated with exposure to the ambient air pollutants nitrogen dioxide (NO2) and particles with a 50% cut-off aerodynamic diameter of 10 μm (PM10) in 7613 adults included in the European Study of Cohorts for Air Pollution Effects (ESCAPE). Effect estimates for FVC were of similar magnitude (for NO2) or larger (for PM10) than those for FEV1. In line with these findings, Forbes et al. [3] showed negative associations of PM10 and NO2 with the level of FEV1 in 40 329 adults included in the Health Survey for England between 1995 and 2001, whereas no significant associations with FEV1/FVC were observed. In 1997, the Swiss Study on Air Pollution and Lung Disease in Adults (SAPALDIA), including 9651 adults, showed negative associations of ambient air pollutants NO2 and PM10 with both FEV1 and FVC [4]. The effect estimates for FVC were stronger than for FEV1 for various pollutants, and this was consistently the case in most subgroups (according to smoking status and respiratory symptoms). Reduced FVC, with FEV1 being normal or reduced to a lesser degree than FVC, suggests restrictive rather than obstructive lung disease (in which FEV1 specifically is reduced, resulting in a low FEV1/FVC ratio). Thus, findings from several European studies suggest that restrictive rather than obstructive ventilatory patterns associate with long-term low levels of exposure to ambient air pollution. A study with slightly different findings is the German Study on the influence of Air Pollution on Lung Function, Inflammation and Ageing (SALIA), including 2593 women. This study also found negative associations of NO2 and PM10 exposure with both FEV1 and FVC, yet the effects estimates for FEV1 were stronger than for FVC, and consequently there were small significant negative associations with the FEV1/FVC ratio [5]. A review article concluded that despite biological plausible mechanisms, there is suggestive, but not conclusive evidence that chronic exposure to air pollution is associated with the prevalence and incidence of chronic obstructive pulmonary disease (COPD), a disease characterised by airway obstruction [6]. Thus far, no studies have focused explicitly on whether air pollution exposure is associated with obstructive or restrictive ventilatory patterns. Exposure to ambient air pollution is associated with restrictive ventilatory patterns http://ow.ly/QWbD30236J1

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.

Age at puberty and risk of asthma: A Mendelian randomisation study

Background Observational studies on pubertal timing and asthma, mainly performed in females, have provided conflicting results about a possible association of early puberty with higher risk of adult asthma, possibly due to residual confounding. To overcome issues of confounding, we used Mendelian randomisation (MR), i.e., genetic variants were used as instrumental variables to estimate causal effects of early puberty on post-pubertal asthma in both females and males. Methods and findings MR analyses were performed in UK Biobank on 243,316 women using 254 genetic variants for age at menarche, and on 192,067 men using 46 variants for age at voice breaking. Age at menarche, recorded in years, was categorised as early (<12), normal (12–14), or late (>14); age at voice breaking was recorded and analysed as early (younger than average), normal (about average age), or late (older than average). In females, we found evidence for a causal effect of pubertal timing on asthma, with an 8% increase in asthma risk for early menarche (odds ratio [OR] 1.08; 95% CI 1.04 to 1.12; p = 8.7 × 10−5) and an 8% decrease for late menarche (OR 0.92; 95% CI 0.89 to 0.97; p = 3.4 × 10−4), suggesting a continuous protective effect of increasing age at puberty. In males, we found very similar estimates of causal effects, although with wider confidence intervals (early voice breaking: OR 1.07; 95% CI 1.00 to 1.16; p = 0.06; late voice breaking: OR 0.93; 95% CI 0.87 to 0.99; p = 0.03). We detected only modest pleiotropy, and our findings showed robustness when different methods to account for pleiotropy were applied. BMI may either introduce pleiotropy or lie on the causal pathway; secondary analyses excluding variants associated with BMI yielded similar results to those of the main analyses. Our study relies on self-reported exposures and outcomes, which may have particularly affected the power of the analyses on age at voice breaking. Conclusions This large MR study provides evidence for a causal detrimental effect of early puberty on asthma, and does not support previous observational findings of a U-shaped relationship between pubertal timing and asthma. Common biological or psychological mechanisms associated with early puberty might explain the similarity of our results in females and males, but further research is needed to investigate this. Taken together with evidence for other detrimental effects of early puberty on health, our study emphasises the need to further investigate and address the causes of the secular shift towards earlier puberty observed worldwide.

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.

No association between DNA methylation and COPD in never and current smokers

Introduction Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory lung disease with cigarette smoke as the main risk factor for its development. Since not every smoker develops COPD, other factors likely underlie differences in susceptibility to develop COPD. Here, we tested if DNA methylation may be such a factor by assessing the association between DNA methylation levels and COPD in never and current smokers from the general population. Methods For the current study, 1561 subjects were non-randomly selected from the LifeLines cohort study. We included 903 never smokers and 658 current smokers with and without COPD, defined as pre-bronchodilator forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) <70%. Subsequently, we performed robust regression analysis on whole blood DNA methylation levels of 420 938 CpG sites with COPD as outcome. Results None of the CpG sites in both the never and the current smokers were genome-wide significantly associated with COPD. CpG site cg14972228 annotated to SIPAL3 was most significant (p=5.66×10−6) in the never smokers, while CpG site cg08282037 annotated to EPS8L1 was most significant (p=1.45×10−5) in the current smokers. Conclusion In contrast to a previous, smaller study, we did not observe any significant association between DNA methylation levels and the presence of COPD, independent of smoking status. Apparently, DNA methylation studies are highly variable.

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.