No convincing association between genetic markers and respiratory symptoms: results of a GWA study
Zeng et al. Respiratory Research
No convincing association between genetic markers and respiratory symptoms: results of a GWA study
Xiang Zeng 0 1
Judith M. Vonk 0 1
Kim de Jong 0 1
H. Marike Boezen 0 1
0 Groningen Research Institute for Asthma and COPD (GRIAC), University of Groningen, University Medical Center Groningen , 1 Hanzeplein, Groningen 9700RB , The Netherlands
1 Department of Epidemiology, University of Groningen, University Medical Center Groningen , 1 Hanzeplein, Groningen 9700RB , The Netherlands
Background: Respiratory symptoms are associated with accelerated lung function decline, and increased hospitalization and mortality rates in the general population. Although several environmental risk factors for respiratory symptoms are known, knowledge on genetic risk factors is lacking. We aim to identify genetic variants associated with respiratory symptoms by genome-wide association (GWA) analyses. Methods: We conducted the first GWA study on cough, dyspnea and phlegm among 7,976 participants in the LifeLines I cohort and used the LifeLines II cohort (n = 5,260) and the Vlagtwedde-Vlaardingen cohort (n = 1,529) for replication. Results: We identified 50 SNPs that were assessed for replication. Rs16918212, located in the alpha-2-macroglobulin pseudogene 1 (A2MP1), was associated with cough in both the identification (odds ratio (OR) = 0.72, p = 5.41 × 10−5) and the meta-analyzed replication cohorts (OR = 0.83, p = 0.033). No other significant replicated associations were found. Conclusions: Given that only 1 out of 50 SNPs showed significant replication (i.e. 2%) we conclude that we did not find a convincing association between genetic markers and respiratory symptoms. Since, environmental exposures are important risk factors for respiratory symptoms, the next step is to perform a genome-wide interaction (GWI) study to identify genetic susceptibility loci for respiratory symptoms in interaction with known harmful environmental exposures.
GWAS; Genetic; Respiratory symptoms; General population; cohorts
The presence of respiratory symptoms, such as chronic
cough, dyspnea and phlegm, is associated with lower
lung function [1, 2] and with mortality due to several
causes of death [3–5]. Respiratory symptoms have been
regarded as important markers of accelerated lung
function decline [6, 7] and development of asthma .
It is known that cigarette smoking , allergy [10, 11],
air pollution [12, 13] and occupational exposures [14, 15]
are risk factors for respiratory symptoms. However, not all
exposed subjects develop respiratory symptoms, which
suggests that a genetic component may be involved in the
development of respiratory symptoms. Previous studies
reported associations between respiratory symptoms and
specific genetic loci using candidate gene studies [16, 17].
To date, only one genome-wide association (GWA) study
has investigated genetic susceptibility of respiratory
symptoms (i.e. Chronic mucus hyper-secretion) . Genetic
susceptibility to develop respiratory symptoms such as
cough, dyspnea, and phlegm has not been studied up until
now using GWA methods.
In the current study, we conducted several GWA
analyses, i.e. on cough, dyspnea and phlegm, in 7,976
Caucasians of Dutch descent from the large
populationbased LifeLines I cohort study to identify common genetic
variants associated with respiratory symptoms. We used
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the LifeLines II cohort and the Vlagtwedde-Vlaardingen
cohort to replicate our initial findings.
Genotyped individuals from the first data release of the
LifeLines cohort study (2006–2011, LifeLines I) with full
data on all covariates were included (n = 7,976). The
LifeLines cohort study is a prospective population-based
cohort studying health and health-related behavior of
subjects from the three Northern provinces of the
Netherlands [19, 20].
We included 5,260 subjects from the second data release
from the LifeLines cohort study (2006–2011, LifeLines
II) and 1,529 subjects from the last survey (1989/1990)
from the Vlagtwedde-Vlaardingen cohort [21, 22], a
prospective general population based cohort including
Caucasians of Dutch descent, to replicate our initial findings.
Ethics, consent and permissions
Participants provided written informed consent. The
study was approved by the Medical Ethics Committee of
the University Medical Center Groningen, Groningen,
The Netherlands (ref. METc 2007/152).
Genotyping and quality control
Genome-wide genotyping was performed in the
identification and replication cohorts using IlluminaCytoSNP-12
arrays. The IlluminaCytoSNP-12 is an oligonucleotide
chip designed to have a uniform spacing of markers across
all chromosomes, with the majority of the markers on this
chip reflecting common SNPs: 93% of the 301,232
markers on this chip reflect bi-allelic SNP markers. The
applied genotyping quality control criteria in the LifeLines
cohort and the Vlagtwedde-Vlaardingen cohort have been
described before [19, 20]: Samples with call-rates of less
than 95% were excluded as were samples of
nonCaucasians and first degree relatives. SNPs were excluded
if they had a genotype call-rate < 95%, minor allele
frequency (MAF) < 1%, or a Hardy-Weinberg equilibrium
(HWE) p-value < 10−4. In the LifeLines cohort 227,981
SNPs were included and in the Vlagtwedde-Vlaardingen
cohort 242,926 SNPs were included.
Cough, dyspnea, and phlegm were defined by
standardized questionnaires from the European Community
Respiratory Health Survey (ECRHS) . Cough was defined
as at least one positive answer to the questions: “do you
usually cough first thing in the morning in the winter?” or
“do you usually cough during the day, or at night, in
winter?”. Dyspnea was defined as a positive answer to the
question: “are you troubled by shortness of breath when
hurrying on level ground or walking up a slight hill or
stairs at normal pace?”. Phlegm was defined as at least one
positive answer to the questions: “do you usually bring up
any phlegm from your chest first thing in the morning in
winter?” or “do you usually bring up any phlegm from
your chest during the day, or at night, in winter?”.
The data are presented as median (min-max) for
continuous variables and as frequencies (percentages) for
categorical variables. The GWA analyses on the presence
of the respiratory symptoms cough, dyspnea, and
phlegm were performed using PLINK version 1.07 .
We used an additive genetic model adjusted for age, sex,
and current smoking. SNPs with a p-value < 10−4 in the
identification analysis were taken forward for replication.
Replication analysis was performed by analyzing the two
replication cohorts separately using logistic regression
model in PLINK version 1.07  and subsequently
meta-analyzing effect estimates from both cohorts.
Significant replication was defined as a fixed effect
metaanalysis p-value < 0.05 and an effect estimate in the same
direction as in the identification GWA study. SNP
annotation was performed using HaploReg version 4 (Broad
Demographic characteristics and the prevalence of
respiratory symptoms in the study cohorts are summarized
in Table 1. In the identification cohort LifeLines I, the
median age of subjects was 47 years old, 43% were male,
and 24% were current smokers. The replication cohorts
were comparable with the identification cohort with
Table 1 Characteristics of the subjects included in the
identification (LifeLines I) and replication (LifeLines II and
Age (yrs), median (min-max) 47 (18–88)
48 (20–89) 53 (35–91)
Current smokers, n (%)
Smoking status, n (%)
Respiratory symptoms, n (%)
respect to demographic characteristics. The prevalence
of respiratory symptoms in the LifeLines cohorts and
Vlagtwedde-Vlaardingen cohort varied from 10 to 22%.
The Manhattan plots of the GWAS of cough, dyspnea
and phlegm are shown in Additional file 1: Figures S1,
S2 and S3 respectively. A total of 17 SNPs, 19 SNPs
and 14 SNPs were identified for cough (Table 2), dyspnea
(Table 3) and phlegm (Table 4) in the identification
analyses in LifeLines I, respectively, and taken forward for
replication in LifeLines II and Vlagtwedde-Vlaardingen.
Rs16918212 (OR = 0.72, p = 5.41 × 10−5 in identification;
OR = 0.83, p = 0.033 in replication), located on A2MP1,
was significantly associated with cough in the replication
cohorts with the same direction of effect as in the
identification cohort (Table 2). The replication analyses on
dyspnea and phlegm showed no significant replication
(Table 3 and Table 4).
In addition, we performed GWA analyses on chronic
cough and phlegm (both defined as cough or phlegm for
at least 3 months per year) and found no significant
replication in these analyses either (Additional file 1:
Tables S1 and S2).
To the best of our knowledge, this is the first GWA
study assessing genetic variants associated with cough,
dyspnea, and phlegm. In the identification cohort, we
identified 17, 19 and 14 SNPs associated with cough,
dyspnea and phlegm respectively at a p < 10−4
significance level. In the meta-analysis of two independent
replication cohorts, one association was observed between
cough and rs16918212 located on chromosome 12 in
intron of A2MP1, and no associations with dyspnea and
phlegm were replicated.
The odds ratio for this SNP indicates that carriers of
the A allele have a lower risk to cough than subjects
with the wild type genotype. This SNP is located in an
intron of A2MP1 (alpha-2-macroglobulin pseudogene
1). A2MP1 has been associated with Alzheimer’s
disease . Pseudogenes are genomic DNA sequences
similar to normal genes but non-functional; they have
lost their gene expression in the cell or their ability to
code protein . Some pseudogenes can be functional
when they are transcribed. Increasing evidence suggests
that pseudogenes may have important physiological
A major strength of this study the fact this is the first
GWA study trying to identify genetic susceptibility loci
for cough, dyspnea and phlegm, which included 2
verification samples: one using the same methodology
(LifeLines II) and one using similar methodology
(VlagtweddeVlaardingen) as the discovery sample (LifeLines I). The
respiratory symptoms that we studied were defined based
on the standardized questionnaire of the ECRHS.
27 kb 5′ of ZNF717
126 kb 5′ of NDN
3.9 kb 3′ of TBCA
77 kb 3′ of HINT1
A GWA study has the advantage of being
hypothesisfree. This means that it has the potential of finding new
genes underlying disease phenotypes . However, GWA
studies also have some disadvantages such as the need of
a large study sample, the need for replication, the inability
to address causation, and the inability to investigate rare
genetic variants .
A limitation of our study might be the fact that we
used a liberal p-value threshold (p < 10−4) for
identification of SNPs in the identification cohort to keep the risk
of not detecting a true association between genetic
markers and respiratory symptoms low. However, when
we assessed these associations in the replication cohorts,
the total number of significant associations in the
replication meta-analysis is less than expected by chance (i.e.
1 out of the 50 SNPs analyzed for replication (i.e. 2%)
had a p-value < 0.05 and the same direction of effect as
in the identification analysis). In addition, given that
rs16918212 and the A2MP1 gene have not been
associated with lung function impairment or respiratory
diseases we think the association is likely not a true finding.
We therefore conclude that there was no convincing
association between genetic markers and respiratory
symptoms in this study.
The lack of finding a plausible significant association
between SNPs and respiratory symptoms can possibly
be explained by the fact that a respiratory symptom can
be caused by different environmental exposures or can
be a presentation of different underlying diseases with
specific genetic or environmental origins. For example,
cough, can be triggered by smoking, air pollution and
occupational exposures. Susceptibility to these various
exposures may be genetically determined and
susceptibility loci may differ between exposures. In addition, cough
is a common symptom of several chronic respiratory
conditions such as asthma, chronic obstructive pulmonary
disease (COPD), and lung cancer , but cough is also
present in non-respiratory conditions such as heart failure
. Dyspnea is a common symptom not only in patients
with lung and heart diseases, but it is also fairly prevalent
among elderly individuals without apparent pre-existing
We did not find a convincing association between genetic
markers and the presence of respiratory symptoms cough,
dyspnea and phlegm. This lack of association between
genetic variants and respiratory symptoms may possibly
be due to the fact that we did not take the effect of
environmental exposures that give rise to respiratory symptoms
into account. Therefore, the next logical step will be
performing a genome-wide interaction (GWI) study to
identify genetic loci for respiratory symptoms in interaction
with known harmful environmental exposures.
Additional file 1: Manhattan plots and additional analyses on chronic
cough and phlegm. (DOCX 584 kb)
SNP: Single nucleotide polymorphism GWAS, Genome wide association study
This study was funded by the Groningen Research Institute for Drug
Exploration (GUIDE), University Medical Center Groningen, University of
Groningen, the Netherlands. The LifeLines Cohort Study, and generation and
management of GWAS genotype data for the LifeLines Cohort Study is
supported by the Netherlands Organization of Scientific Research NWO
(grant 175.010.2007.006), the Economic Structure Enhancing Fund (FES) of
the Dutch government, the Ministry of Economic Affairs, the Ministry of
Education, Culture and Science, the Ministry for Health, Welfare and Sports,
the Northern Netherlands Collaboration of Provinces (SNN), the Province of
Groningen, University Medical Center Groningen, the University of Groningen,
Dutch Kidney Foundation and Dutch Diabetes Research Foundation. The
Vlagtwedde-Vlaardingen cohort study was supported by the Ministry of Health
and Environmental Hygiene of the Netherlands and the Netherlands Asthma
Fund (grant 187) and the Netherlands Asthma Fund grant no. 3.2.02.51, the
Stichting Astma Bestrijding, BBMRI-NL (Complementiation project), and the
European Respiratory Society COPD research award 2011 to H.M. Boezen.
The sponsors had no role in the study design, data collection, analysis and
interpretation, or in writing and submitting the manuscript.
Availability of data and materials
Please contact author for data requests.
XZ and KdJ performed the statistical analyses and drafted the first version of
the manuscript. HMB designed the study. HMB and JMV supervised the
statistical analyses. JMV, KdJ, XX, XH and HMB interpreted the results and have
written the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Participants provided written informed consent. The study was approved by
the Medical Ethics Committee of the University Medical Center Groningen,
Groningen, The Netherlands (ref. METc 2007/152).
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