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Pathophysiology of asthma: lessons from genetic research
with particular focus on severe asthma
E. Melen1,2 & G. Pershagen1
From the 1Institute of Environmental Medicine and Centre for Allergy Research, Karolinska Institutet; and 2Astrid Lindgren Childrens Hospital,
Karolinska University Hospital, Stockholm, Sweden
Abstract. Melen E, Pershagen G (Institute of Environ-
mental Medicine and Centre for Allergy Research,
Karolinska Institutet; Astrid Lindgren Childrens
Hospital, Karolinska University Hospital, Stock-
holm, Sweden). Pathophysiology of asthma: lessons
from genetic research with particular focus on severe
asthma (Reveiw). J Intern Med 2012; 272: 108120.
There is good evidence that both inherited and envi-
ronmental factors influence the risk of developing
asthma. Only recently, large well-designed studies
have been undertaken with the power to identify the
genetic causes for asthma, and methods developed in
parallel with the Human Genome Project, such as
gene expression and epigenetic studies, have made
large-scale analyses of functional genetics possible.
In this review, we discuss the recent findings from ge-
Introduction
Around 5% of children with asthma suffer from
chronic symptoms and or severe exacerbations de-
spite treatment with a variety of drugs, including high
doses of inhaled corticosteroids [1]. The clinical char-
acteristics of severe asthma in children include
chronic inflammation and subsequent structural
changes of the airways, comorbidities and poor lung
function [25], indicating that genes involved in these
processes may be of particular interest. From twin
studies, it seems that the heritability of asthma may
be as high as 6070%, particularly in preschool chil-
dren [6, 7]. Also, it appears that there is a consider-
able hereditary component with regard to the severity
of asthma [8]. Results from longitudinal studies sup-
port the role of hereditary factors as severe childhood
asthma often continues into adulthood [9]. No single
gene has yet been identified that can explain the
majority of asthma cases or aid the prediction of dis-
ease prognosis. Only recently, large well-designed
studies have been undertaken with the power to iden-
tify the genetic causes for asthma, and methods
developed in parallel with the Human Genome Project
108 2012 The Association for the Publication of the Journal of Internal Medicine
doi: 10.1111/j.1365-2796.2012.02555.x
netic and genomic research studies of asthma, partic-
ularly severe asthma, and highlight specific genes for
which there are multiple lines of evidence for involve-
ment in asthma pathogenesis. Bio-ontologic enrich-
ment analyses of the most recently identified asthma-
related genes point to attributes such as molecular
and signal transducer activity and immune system
processes, which indicates the importance of immu-
noregulation and inflammatory response in the path-
ogenesis of asthma. Finally, we discuss how genetic
and environmental factors jointly influence asthma
susceptibility and summarize how the results may
increase understanding of the pathophysiology of
asthma-related diseases.
Keywords: asthma, children,genetics, genomics, severity.
have made possible large-scale analyses of functional
genetics, such as gene expression and epigenetic
studies.
Here we review the recent findings from genetic and
genomic research studies of asthma, and severe asth-
ma in particular, and highlight specific genes for
which there are several lines of evidence for involve-
ment in asthma pathogenesis. In addition, we dis-
cuss how genetic and environmental factors jointly
influence asthma susceptibility and summarize how
the results may increase understanding of the patho-
physiology of asthma-related diseases.
Insight from genetic studies of asthma before the era of genome-wide
association studies
The first linkage analyses for genes involved in asth-
ma susceptibility and IgE regulation initiated in the
early 1990s appeared promising [10]; however, the
first candidate gene (ADAM 33) was not identified un-
til 2002 [11]. ADAM33 belongs to a family of metallo-
proteinases and is predominantly expressed in cells
in the airways, such as fibroblasts and smooth
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma

muscle cells. Because of its broad involvement in cell Number of studies

with posive associaon
activation and signalling, it has been suggested that 12

ADAM33 is involved in the remodelling process that >10 Studies

10
may occur in asthma patients with long-standing air-
way inflammation. Single nucleotide polymorphisms 8 610 Studies
(SNPs) in ADAM33 have been shown to influence
6
baseline lung function as well as asthma and bron-
chial hyperresponsiveness, but do not appear to in- 4
25 Studies

crease the risk of allergy [12]. A meta-analysis of the

association between ADAM33 SNPs and asthma 2 1 Study
showed an overall 46% risk increase of a particular
0
Gene names
SNP (ST+7) [13]. HAVCR1
NOS3
ADRB2
CCL11
IFN-
IL10
NPSR1
PAFAH
IL4R
GSTP1
IL4
TNF
CCL24 IL12b PHF11 IL13
CCL5 INPP4A PTGDR ORMDL3
To date, eight genes (DPP10, PHF11, GPRA NPSR1, CD14 IRAK-3 TBXA2R ADAM33
CHI3L1 ITGB3 TGFB1 FCER1B
HLA-G, CYFIP2, IRAK-M IRAK3, OPN3 and PCDH1) CTLA4 LTA TLR10
CX3CR1 MYLK TLR4
have been identified by positional cloning, which has CYSLTR2 NAT2 TLR9
DPP10 NOD1 UGB (CC10) Adapted from Weiss ST et al,
led to increased knowledge of the pathophysiology of EDN1 NPPA VDR Curr Opin Genet Dev 2009;19:279282

asthma and allergy [1421]. Much work is still ongo-

ing to elucidate the role of these genes. In genetic
association studies, replication of results in indepen-
dent data sets is generally considered good evidence
for the identification of a true disease-susceptibility
gene. Several reviews of asthma genetics in recent
years have been published [2225]. In 2006, it was
concluded that the total number of genes that con-
tribute to susceptibility to asthma or atopy may ex-
ceed 100 [22]. Based on updated data from 2008,
asthma-susceptibility genes were classified into
genes with four main functions: (i) innate immunity
and immunoregulation (e.g. CD14, HLA genes and
TLR4), (ii) T-helper 2 (Th2)-cell differentiation and
activity (e.g. IL4 IL4R, IL13 and FCER1B), (iii) epithe-
lial biology and mucosal immunity (e.g. CCL-genes
and FLG), and (iv) lung function, airway remodelling
and asthma severity (e.g. ADRB2 and TNF) [23]. Using
more stringent criteria for association, that is, associ-
ation with the asthma phenotype in at least one study
with more than a total of 300 subjects (150 cases and
150 controls) and one other population as well as rep-
lication with the same SNP, only 43 asthma candidate
genes reported until 1 July 2008 were identified
(Fig. 1) [24].
Severe asthma
Few genetic studies of representative problematic se-
vere asthma cases have been performed, particularly
in children, and the causes for severe therapy-resis-
tant childhood asthma are still poorly understood.
Many studies have, however, addressed asthma
severity as exacerbations, need for rescue therapy or
lung function deterioration in patients with mild-to-
moderate asthma [22]. Whether there are specific
genetic variants that contribute to the development of
Fig. 1 Studies showing positive gene association(s) with
asthma, using stringent inclusion criteria for association (at
least two populations included), published until 1 July 2008.
ORMDL3 from the first asthma GWAS (see text for further
details) was also included. Adapted from the reference [24].
severe, therapy-resistant asthma, but not exacer-
bations in patients with usually mild-to-moderate
asthma, remains to be elucidated. A limited number
of genes have been specifically associated with severe
asthma, amongst them IL4RA. In two well-character-
ized American cohorts of patients with severe asth-
ma, two SNPs coding for amino acid substitutions
(E375A and Q551R) were associated with severe
exacerbations and lower lung function measured as
forced expiratory volume in 1s, FEV1 [26]. The results
were further supported by an increase in tissue mast
cell numbers and higher levels of specific IgE bound
to mast cells in carriers of the risk alleles. In addition
to numerous studies linking IL4RA polymorphisms
to asthma per se [22], it has been suggested that
genetic variation in IL4 (C-589T) is a risk factor for
life-threatening asthma attacks [27]. Furthermore,
CTTN, PHF11 and TNF are examples of genes that
have been associated with severe asthma [28].
Genome-wide association studies of asthma and asthma-related traits
In 2007, the first genome-wide association study
(GWAS) of asthma (994 patients with childhood-on-
set asthma and 1243 nonasthmatic subjects) was
published, and genetic variants regulating ORMDL3
expression on chromosome 17q21 were identified as
strong determinants of asthma susceptibility
(P < 10)11) [29]. ORMDL3 was initially identified in
2002 by sequence comparison with another member

2012 The Association for the Publication of the Journal of Internal Medicine 109
Journal of Internal Medicine, 2012, 272; 108120
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma
of the same family of proteins, ORMDL1 [30]. The OR-
MDL genes encode transmembrane proteins that are
anchored into the endoplasmic reticulum and are be-
lieved to be involved in protein folding. ORMDL genes
show high sequence conservation between many
species, which supports a crucial role for cellular
functions. Specifically, it has been suggested that
ORMDL3 is involved in cellular responses to inflam-
mation and dysregulation of sphingolipid metabo-
lism, but the exact mechanisms of action are still
unclear [31].
The association between ORMDL3 variants and asth-
ma has been confirmed in several large data sets [32
35], including the largest GWAS of asthma to date
with more than 10 000 asthma cases and 16 000
controls (the GABRIEL study) [36]. A meta-analysis
of five published studies of a particular SNP
(rs7216389) in nine populations demonstrated an
odds ratio for asthma of 1.44 in individuals carrying
the risk allele [37]. This SNP was associated with
asthma and ORMDL3 expression with the highest de-
gree of statistical significance in the original GWAS
[29]. Association with increased asthma exacerba-
tions in children despite current medications has
also been reported [38], as well as with increasing
asthma severity in adults [39]. However, the mecha-
nisms underlying the increased risk of exacerbations
are not fully known at present. Analyses stratified by
age of onset suggest that ORMDL3 variants are partic-
ularly associated with childhood-onset asthma [40];
this finding was confirmed in the GABRIEL study
[36]. Clinical studies suggest that childhood and
adult asthma differ with regard to a number of char-
acteristics, including sex preponderance, airflow
obstruction, sensitization and structural changes of
the airways, although remodelling may already occur
in children with severe asthma [4143].
Although the genetic association between ORMDL3
and childhood asthma seems very robust across
studies and populations, the disease risk association
with a single genetic variant is modest as exemplified
by the above-mentioned meta-analysis. This is ex-
pected for a complex disease such as asthma, but lim-
its the use of single variants in genetic testing and
prediction algorithms. In addition, several variants in
the chromosome 17q21 region, which also includes
the GSDML and ZPBP2 genes, are associated with
asthma but the mechanisms underlying the risk of
asthma associated with these variants have not been
fully elucidated. Gene expression studies have, how-
ever, given new insights into the genetic regulation of
this region (see later for discussion).
110 2012 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2012, 272; 108120
Data regarding DNA variants, gene expression and
protein levels were all used in a study that lead to
the identification of the CHI3L1 gene as a new asth-
ma-susceptibility locus [44]. The CHI3L1 gene en-
codes for YKL-40, a protein found in the lungs and
circulation that has been suggested to be a new bio-
marker for severity of asthma [45]. Rather than
using asthma as the phenotype, Ober et al. con-
ducted a GWAS using serum YKL-40 levels as the
outcome. A promoter SNP (-131 C G) in CHI3L1
was found to be strongly associated with elevated
YKL-40 levels (P = 1.1 10)13), as well as with asth-
ma and lung function. YKL-40, which is a chitinase-
like protein, is produced by various inflammatory
cells and may have an important role in asthma
development and disease deterioration. The associ-
ations between CHI3L1 and asthma have been repli-
cated in other populations, but further studies on
asthma and asthma-related phenotypes are war-
ranted [24].
Recent asthma GWASs and bio-ontologic enrichment analysis
As of January 2012, the results of 21 GWASs of
asthma or asthma-related traits had been published
and markers in 36 genes had been identified with a
commonly used cut-off of P 10)6 for association
(Table 1) [46]. To further explore the biology of the
genes identified in GWASs, we performed gene ontol-
ogy (GO) enrichment analysis using the Database
for Annotation, Visualization and Integrated Discov-
ery (DAVID) [47, 48]. In terms of bio-ontologic
enrichment, the genes shown to be associated with
asthma in GWASs were enriched (P < 0.05) for onto-
logical attributes such as molecular signal trans-
ducer activity (13 of the 36 identified genes: HLA-
DQB1, IL18R1, IL2RB, IL1RL1, SMAD3, RORA, IL6R,
HLA-DQA2, NOTCH4, GAB1, HLA-DPB1, HLA-DOA
and HLA-DRA) and immune system process (11 of
the 36 genes: HLA-DQB1, IL18R1, IL1RL1, NOTCH4,
SMAD3, IL13, IL6R, HLA-DPB1, HLA-DOA, HLA-
DQA2 and HLA-DRA). The enrichment results were
largely driven by two groups of genes represented by
the MCH class II genes and cytokine receptor activity
(IL18R1, IL2RB, IL1RL1 and IL6R), which indicates
the importance of immunoregulation and inflamma-
tory response in the pathogenesis of asthma. The
identified interleukin (IL) receptors, as well as the
IL33 loci confer involvement of the IL gene family
and their receptors in asthma. IL33 with its receptor
IL1RL1 belongs to the IL1 family of cytokines that
are known to be expressed on epithelial cells and are
involved in the host response to certain environmen-
tal and infectious stimuli [49]. Severe asthma has
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma
Table 1 Significant bio-ontologic attributes of genes identified in GWASs of asthma
Category Ontological attribute
GOTERM_MF_1 GO:0060089 Molecular transducer activity HLA-DQB1, IL18R1, IL2RB, IL1RL1, SMAD3, RORA,
GOTERM_MF_2 GO:0004871 Signal transducer activity
GOTERM_BP_1 GO:0002376 Immune system process
GOTERM_MF_3 GO:0004872 Receptor activity
GOTERM_BP_2 GO:0006955 Immune response
GOTERM_BP_2 GO:0019882 Antigen processing
and presentation
GOTERM_CC_3 GO:0042611 MHC protein complex
GOTERM_CC_4 GO:0042613 MHC class II protein complex
GOTERM_MF_5 GO:0032395 MHC class II receptor activity
GOTERM_MF_3 GO:0019838 Growth factor binding
GOTERM_MF_3 GO:0019955 Cytokine binding
GOTERM_MF_4 GO:0004896 Cytokine receptor activity
KEGG_PATHWAY hsa05310: Asthma
KEGG_PATHWAY hsa05330: Allograft rejection
hsa04940: Type I diabetes mellitus
hsa05320: Autoimmune thyroid disease
hsa05416: Viral myocarditis
hsa05322: Systemic lupus erythematosus
In total, 36 genes were identified in recent GWASs. Genes not characterized by the ontological attributes listed in the table
include BTNL2, CDK2, CRCT1, CTNNA3, DENND1B, GSDMA, GSDMB, IKZF4, IL33, LRRC32, PBX2, PCDH20, PDE4D, PRKG1,
PYHIN1, RAD50, SCG3, SLC22A5, SLC30A8, TLE4 and TSLP.
been particularly studied in two GWASs, both of
which indicated the importance of Th2-like genes in
the locus on chromosome 5q containing RAD50 and
IL13 [50], and TSLP on the same chromosome [36].
In addition, association with HLA-DQB1 was ob-
served [50]. Using emergency department visits or
hospitalizations as the definition of a severe asthma
exacerbation in the CAMP study, a combination of
160320 SNPs were found to predict exacerbation
with an area under the curve (AUC) of 0.66 [51]. For
a complex trait such as severe asthma, prediction
has proven to be very challenging, and the AUC in
this study shows that genetic analyses can improve
our diagnostic tools, although much work remains
before clinical testing can become a reality. Amongst
the top 160 SNPs identified in the CAMP study, one
(rs10496476) is located within DPP10, which was
identified as an asthma-susceptibility gene by posi-
tional cloning [14].
Identified genes represented by each term
IL6R, HLA-DQA2, NOTCH4, GAB1, HLA-DPB1,
HLA-DOA, HLA-DRA
HLA-DQB1, IL18R1, IL1RL1, NOTCH4, SMAD3,
IL13, IL6R, HLA-DPB1, HLA-DOA, HLA-DQA2, HLA-DRA
HLA-DQB1, IL18R1, IL2RB, IL1RL1, NOTCH4, IL6R,
HLA-DPB1, RORA, HLA-DOA, HLA-DQA2, HLA-DRA
HLA-DQB1, IL18R1, IL1RL1, SMAD3, IL13, IL6R,
HLA-DPB1, HLA-DOA, HLA-DQA2, HLA-DRA
HLA-DQB1, HLA-DPB1, HLA-DOA, HLA-DQA2, HLA-DRA
IL18R1, IL2RB, IL1RL1, IL6R
HLA-DQB1, IL13, HLA-DPB1, HLA-DOA, HLA-DQA2, HLA-DRA
HLA-DQB1, HLA-DPB1, HLA-DOA, HLA-DQA2, HLA-DRA
The suggested Kyoto Encyclopedia of Genes and Ge-
nomes (KEGG) pathways for this group of 36 asthma
genes include asthma represented by the MCH class
II genes and IL13 (Table 1). Thus, only six of the 36
genes were captured by the KEGG terms, and the
same MCH genes were also captured by several other
autoimmune diseases such as type I diabetes, auto-
immune thyroid disease and systemic lupus erythe-
matosus. This indicates that shared genetics is likely
to be of importance for several diseases related to
inflammation and autoimmunity. It is well known
that the MCH genes are involved in these diseases,
and shared genetic effects have also been implicated
from previous studies on network analyses of com-
plex diseases [52]. However, most of the genes identi-
fied in GWASs to be associated with asthma were not
captured by GO or KEGG pathway terms, suggesting
that our current understanding of the role of these
genes in asthma pathogenesis is incomplete. For
2012 The Association for the Publication of the Journal of Internal Medicine 111
Journal of Internal Medicine, 2012, 272; 108120
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma
instance, genes in the ORMDL3 locus, currently con-
sidered to be the strongest locus for childhood asth-
ma, were not captured by the enrichment analysis.
This highlights the limitations of the use of current
databases and bioinformatics tools because informa-
tion regarding new genes with unknown or only
partly known functions is inconsistent with previous
knowledge of human physiology and disease patho-
genesis. This does not mean that the encoded gene
products and pathways implied by these new genes
are not relevant for asthma pathogenesis. On the con-
trary, it suggests that substantial efforts are needed
to clarify the role of these new genes for asthma and
related diseases. A review and detailed description of
GWAS-identified asthma-associated genes reported
until 2010 and their involvement in asthma patho-
genesis was recently published [31].
Whole-genome sequencing
By design, rare variants or mutations are not cap-
tured by current GWAS chips and thus cannot be
evaluated in these studies. It has been suggested that
rare variants are important for several complex dis-
eases [53], but to date, few studies have addressed
the role of rare variants in the pathogenesis of asth-
ma. However, studies of, for example, FLG, IL4,
IL12RB1 and TACI point to a potentially important
role for rare variants in asthma pathogenesis [5457].
There is currently a very rapid development in
sequencing technologies, usually referred to as next-
generation sequencing (NGS), in terms of accuracy,
genetic cover, speed and costs [58]. A major advan-
tage of whole-genome sequencing is the possibility of
detecting genetic variants other than SNPs, including
rare variants, insertions deletions, inversions and
copy number variations (CNVs). NGS will in the near
future offer large-scale sequencing capabilities in ge-
netic studies as well as clinical settings. The 1000 Ge-
nomes Project (http://www.1000genomes.org/) is
the first project to sequence the genomes of a large
number of people with the aim of providing a compre-
hensive map of human genetic variation [59].
Genomic studies of asthma
Gene expression is one of many mechanisms that
may affect the protein level and its influence on cellu-
lar functions. Variation in gene expression is there-
fore vitally important for normal cellular events as
well as for processes related to disease development.
The DNA sequence is the template for mRNA tran-
scription (i.e. gene expression), which will determine
112 2012 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2012, 272; 108120
the transcript sequence and ultimately the protein
product (Fig. 2). However, there are a number of regu-
latory mechanisms in cells that will influence the
transcription rate (e.g. binding of transcription fac-
tors to the promoter region and epigenetic changes),
transcript sequence (posttranscriptional splicing or
alternative polyadenylation) and translation into pro-
tein [60]. Regulatory, nonprotein-coding RNAs have
attracted much attention recently and are believed to
have important roles in transcriptional and posttran-
scriptional regulation [61]. In addition, environmen-
tal stimuli may be potent triggers of expression of spe-
cific genes. Thus, determining the DNA sequence of a
particular region will only provide the underlying ba-
sis for biological functions that may be responsible
for disease development and not the whole picture.
By studying gene expression and protein characteris-
tics, we will obtain better functional information
about processes related to health and disease. Gene
expression is in many cases tissue specific, which is
important to consider when studies are compared.
On the other hand, analyses of tissues directly af-
fected by the disease (e.g. airway epithelial or smooth
muscle cells from patients with asthma) may give
valuable insights into the pathophysiology of the tar-
get organ.
Examples from candidate expression studies
Similar to candidate gene studies of asthma, several
candidate expression studies of asthma have also
been published during the last 15 years. Targeted
PCR primers for a specific region or gene are usually
designed, and the region of interest is amplified and
the amount of RNA quantified. For example, chronic
severe asthma has been shown to be accompanied by
Transcription
DNA (antisense) 3T A A T T T G A T5
mRNA (sense) 5A U U A A A
Codon
Translation
Amino acid Protein
Regulatory effects
- microRNA
- siRNA etc
Fig. 2 Transciption of messenger RNA (mRNA) from DNA in
the cell nucleus, and further translation into protein which
takes place in the cytoplasmic ribosomes. Regulatory effects
of noncoding RNA molecules such as microRNA and siRNA
are also indicated.
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma
an upregulation of TNF expression in peripheral
blood cells and high levels of tumour necrosis factor
alpha (TNF-a) in bronchoalveolar lavage (BAL) fluid
[62, 63]. At the gene level, the TNF promoter G-308A
in particular has been associated with enhanced in vi-
tro transcription and increased TNF-a levels in white
blood cells [64]. From a recent meta-analysis, it was
concluded that the -308A allele confers a significant
risk of asthma, which links TNF with asthma at the
DNA, RNA and protein levels [65].
One of the most studied candidate genes, IL13, for
which association with asthma and allergy has been
well replicated, has also been the focus of substantial
functional studies, especially with regard to its broad
involvement in allergic inflammation [66]. For exam-
ple, it has been proven that the IL13 promoter SNP-
1112CT has functional effects in that enhanced pro-
moter activity and increased IL13 transcription are
seen in human and mouse Th2 cells [67]. The same
SNP has also been associated with both asthma and
allergy [22], and IL13 (IL4 IL13 locus) was the only
gene that showed associations with both asthma and
an allergy-related trait (total IgE) in a GWAS [36]. Thy-
mic stromal lymphopoietin (TSLP) is a cytokine re-
leased from airway epithelial cells in response to
pathogens or inflammatory cytokines such as IL13.
Two studies support its involvement in the pathogen-
esis of severe asthma: a functional study showed that
TSLP protein expression was significantly increased
in airway epithelium of patients with asthma, partic-
ularly those with severe disease [68], and results of
the GABRIEL study suggested an association be-
tween TSLP SNPs and severe asthma [36].
IL33 is another example of a member of the IL family
for which evidence of involvement in asthma patho-
genesis has been provided from various sources in re-
cent years [49]. In addition to being identified in re-
cent GWASs, functional studies suggest that IL33
may have a key role in host responses to stimuli such
as exposure to allergens, air pollutants and respira-
tory viruses that may cause damage to the airway
through IL33 activation and inflammation triggering.
IL33 may also catalyse sensitization to allergens
through dendritic cell activation and Th2-mediated
pathology. Furthermore, IL33 is upregulated in air-
way smooth muscle cells, which are key cells in the
regulation of bronchial responsiveness and airway
tonus, from patients with severe asthma, suggesting
an involvement in therapy-resistant asthma [69].
In several positional cloning studies related to asth-
ma, gene expression patterns have been used to sup-
port the findings of genetic associations. For exam-
ple, expression of GPRA NPSR1 was found to be
markedly increased in bronchial biopsies from pa-
tients with asthma compared with healthy controls,
and Gpra Npsr1 mRNA was significantly upregulated
in lung tissue from sensitized mice compared with
control mice [17]. Neuropeptide S (NPS) activates sig-
nalling through its receptor NPSR1; the effect of
receptor activation on gene expression has been
characterized in cell lines using microarray analysis
[70]. This revealed a set of 300 genes with altered
expression. One of the upregulated genes, showing a
robust >4-fold change, was Tenascin C (TNC). Subse-
quently, it was shown that experimental NPS stimu-
lation of NPSR1-transfected cells upregulated TNC
mRNA in a dose-dependent manner [71]. Epistatic ef-
fects between NPSR1 and TNC variants also altered
the risk of allergic disease, and this interaction high-
lights the complex interplay between several genes
and their products for the development of asthma
and allergy. For another positionally cloned gene,
ADAM33, increased protein levels in BAL fluid from
patients with severe asthma supports its role in dis-
ease development [72]. Expression of ADAM33 in epi-
thelial and fibroblast cells from bronchial biopsy
specimens has been shown to be controlled by epige-
netic mechanisms (methylation status of a regulatory
CpG island within the ADAM33 promoter) [73]. Of
importance, these results demonstrate that (i) gene
expression can be very cell specific even in adjacent
cell tissues both relevant for a certain disease (e.g.
expression of ADAM33 in fibroblasts but not epithe-
lial cells in asthma) and (ii) expression can be tightly
controlled by epigenetic mechanisms as low levels of
methylation were detected in fibroblasts that express
ADAM33, whereas epithelial cells that do not express
ADAM33 showed a very high level of promoter methyl-
ation (see further discussion of epigenetics later).
Whole-genome expression
The transition from a candidate gene to a whole-gen-
ome approach for DNA variants has been accompa-
nied, or even preceded by, similar development in
expression analysis. The method of whole-genome
expression analysis has been available for more than
a decade, enabling an unbiased view of upregulated
and downregulated genes in relation to a specific phe-
notype or disease. However, there have been rela-
tively few studies of human asthmatic tissue samples
and most have been hampered by small sample sizes.
Using a global approach, hundreds or thousands
of genes may be differentially expressed between
cell types or between individuals with or without a
2012 The Association for the Publication of the Journal of Internal Medicine 113
Journal of Internal Medicine, 2012, 272; 108120
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma
particular disease. These results are often compli-
cated to interpret at an individual gene level, and net-
work or pathway approaches are therefore attractive
options. This field is rapidly evolving under the um-
brella term systems biology [74]. Within an ongoing
large European collaborative project (MeDALL), ef-
forts are being made to bridge the gap between func-
tional genetic research and systems biology with clin-
ical and epidemiological research to increase our
understanding of the causes for asthma and allergic
disease [75].
Severe asthma exacerbations in children are often
triggered by viral infections, but the cellular and
molecular mechanisms associated with these exacer-
bations are poorly understood. Using a genomics-
based approach involving microarray profiling of
peripheral blood mononuclear cells collected during
an acute exacerbation and afterwards during conva-
lescence, gene expression patterns were studied in a
cohort of asthmatic children [76]. The results of a sim-
ilar study in adults have also been published recently
[77]. The genes identified as differentially expressed
during exacerbation and convalescence in children
comprised genes associated with (i) arachidonic
acid prostaglandin metabolism, (ii) leucocyte migra-
tion, (iii) innate immunity, (iv) adaptive immunity,
(v) the complement coagulation cascade, and (vi)
inflammation. One of the most highly upregulated
genes during acute exacerbation was CCR2, previ-
ously shown to play an important role in trafficking of
dendritic cells and monocytes to the lung during air-
way inflammation. A key finding was the upregula-
tion of another well-known gene involved in allergic
responses and regulation of IgE levels, FceR1 (the
high-affinity IgE receptor), which suggests that respi-
ratory viral infections in children may initiate an ato-
py-dependent cascade of signalling events. FceR1 has
also been significantly associated with total IgE levels
in peripheral blood in two GWASs [36, 78].
Using genome-wide expression arrays, the tran-
scriptomes of asthmatic children from Sweden were
characterized, and a difference in gene expression be-
tween severe, therapy-resistant asthma and mild
asthma could be observed [79]. Traditional immune-
related functions, such as T-cell receptor signalling
and natural killer cell-mediated cytotoxicity, were in
this study found to be upregulated in children with
controlled asthma, but such patterns were not ob-
served in those with severe asthma. Instead, a signifi-
cant enrichment and upregulation of bitter taste
receptors (TAS2Rs) was found, which highlighted
new pathways contributing to therapy-resistant
114 2012 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2012, 272; 108120
asthma. Until recently, theTAS2Rs were not known
to have any clear role in asthma. However, it has been
found that the TAS2Rs are expressed in human air-
way smooth muscle and that activation of these
receptors causes relaxation of smooth muscles and
dilatation of airways. Inhaled bitter tastants also
caused decreased airway obstruction in a mouse
model of asthma [80]. The bronchodilating effects of
TAS2R agonists have been replicated in other airway
models [81]. Based on these findings, the expression
of TAS2Rs in peripheral blood cells in patients with
severe asthma is currently under further examina-
tion to clarify the role of these receptors in asthma.
Figure 3 shows a simplified schematic diagram of our
current understanding of the genetic and genomic
contributions to severe asthma, where association at
the DNA or RNA level has been established. For some
of the associated genes, there is also evidence of
altered protein secretion in cells with a key role in
asthma, as indicated in Fig. 3.
Moffatt et al. [29] have provided an illustrative exam-
ple of a successful combination of association and
gene expression analyses for ORMDL3. In addition to
the GWAS analysis showing strong association to the
chromosome 17q21 region, global gene expression in
Epstein-Barr virus-transformed lymphocytes (EB-
VLs) from 378 asthmatic children and their siblings
was measured. The same subjects had been geno-
typed with a GWAS platform (Illumina). EBVLs repre-
sent the B-cell lineage and have been shown to have
direct relevance to asthma. Transcripts in one gene,
ORMDL3, were strongly (P < 10)22) associated with
exactly the same SNPs as found in the GWAS. It was
further estimated that the disease-associated SNPs
(also known as expression quantitative trait loci, eQ-
TLs, when expression is affected) accounted for
29.5% of the variance of ORMDL3 expression.
The fact that several markers at the chromosome
17q21 locus were associated with asthma in the origi-
nal and follow-up studies, along with the strong link-
age disequilibrium between these markers, makes it
impossible to identify one specific variant that is more
important than any others as the definitive risk vari-
ant. In addition, ORMDL3 has been associated with
autoimmune diseases such as Crohns disease and
type 1 diabetes, which suggests that this locus may
harbour variants that exert transcriptional control
with broad effects [82, 83]. Verlaan et al. [84] under-
took a series of in-depth functional experiments in
lymphoblastoid cell lines and demonstrated that the
genetic variants associated with asthma act over a
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma

Airway
Protein sectretion:
TNF-
Epithelial cells TSLP

ADAM33
IL33

Smooth muscle cells

Transcription

Association with DNA Altered gene expression

variants in: (RNA level) in target cells:
Fig. 3 Epithelial and smooth CTTN IL4 ADAM33
muscle cells lining the bronchial IL4R IL13 IL4R
tree and involvement in the HLA-DQB1 ORMDL3 IL33
PHF11 RAD50 PHF11
pathogenesis of severe asthma TNF
TNF TSLP
at the DNA, RNA and protein TAS2Rs
levels. TSLP

large genomic region and affect the expression of not rs12936231 G/C SNP Effect on gene expression
only ORMDL3 but also the nearby ZPBP2 and GSDMB
ZPBP2 GSDMB ORMDL3
genes. CTFC
G

It has been suggested that one particular SNP

(rs12936231, intronic) in the ZPBP2 gene is the key C

regulator in the region. This SNP alters the sequence

of putative binding sites for CTCF (a DNA insulator
protein). When CTCF binds to insulator DNA ele-
ments it may block enhancers from activating their
target genes via formation of chromatin loops. In the
study by Verlaan et al., it was hown that the G allele of
rs12936231 specifically bound CTCF, whereas the C
allele did not. In turn, the presence of the G allele (and
CTFC) was associated with lower GSDMB and OR-
MDL3 and higher ZPBP2 expression (Fig. 4). This phe-
nomenon is known as allelic expression, and refers to
the potential of a variation on one allele to alter the
expression of one or more genes on the same allele.
Variation of gene expression between alleles has been
shown to be fairly common in the human genome,
with potentially large contributions to human vari-
ability [85].
It has been suggested that common genetic mecha-
nisms underlie lung morphogenesis and pathogene-
sis of respiratory diseases, which is of clear relevance
to both asthma and chronic obstructive lung disease.
In this setting, transcriptome analyses have also been
used to broaden our understanding of the genetic
influence on normal and pathological development of
certain tissues (e.g. lung development) [86]. Amongst
Fig. 4 Allelic expression of ZPBP2, GSDMB and ORMDL3
genes owing to a G C nucleotide change (rs12936231) that
affects CTFC binding and chromatin remodelling. Adapted
from the reference [84].
the asthma genes identified in GWASs, ROBO1,
RORA, HLA-DQB1, IL2RB and PDE10A have been
found to be differentially expressed during human
lung development [25]. Determinants for normal lung
development are critical not only early in life, but also
for later lung function. These results support a role
for these asthma-susceptibility genes also in normal
lung physiology.
The link between environmental exposure and genetic factors
Geneenvironment interactions
The aim of studies of geneenvironment interactions
is to identify genetic variants that may determine
individual variability in responses to environmental
factors. Ambient air pollution and environmental
tobacco smoke, for example, are known to induce for-
mation of hazardous oxidants and airway inflamma-
tion, and thereby trigger asthma symptoms. There is

2012 The Association for the Publication of the Journal of Internal Medicine 115
Journal of Internal Medicine, 2012, 272; 108120
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma

CH3 example, children with a specific GSTP1 variant

(105Val) have been shown to be at high risk of allergy
5A T C C G A C T A3 and asthma if exposed to high levels of traffic-related
3T A G G C T G A T5 air pollutants compared with children homozygous
for the reference allele (Ile Ile) [95]. These studies
CH3 highlight the complex interactions between genes
and the environment; further investigation is war-
Fig. 5 Methylation (CH3 group) at a CpG site in the DNA ranted, especially in children with well-categorized
sequence. subgroups of severe asthma who may be particularly
sensitive to certain environmental factors.

good evidence that long-term exposure to ambient air

pollutants exacerbates asthma [87]. Some birth co-
hort studies also show association between exposure
and incident asthma [8892]. Whether the effect of
exposure to air pollution is modified by a persons ge-
netic profile to confer increased or decreased risk of
asthma development has been investigated in a lim-
ited number of studies in children. In this context,
genes involved in the anti-oxidative system (e.g. gluta-
thione S-transferase (GST) M1 and P1), the inflamma-
tory response (e.g. TNF) and innate immunity (e.g.
TLR2 and TLR4) have been the particular focus of
studies of asthma and related disorders [9397]. For
Epigenetic studies
Epigenetics can be defined as changes in gene expres-
sion, which cannot be explained by variations in the
underlying DNA sequence. Epigenetic changes may
persist over a number of cell generations, and new
data support inheritance from parents to offspring
[98]. Epigenetic mechanisms include DNA methyla-
tion (binding of a methyl group to the DNA, usually
where cytosine (C) lies adjacent to guanine (G), termed
a CpG site) and chemical modification (acetylation or
phosphorylation) of histones and chromatin struc-
tures (Fig. 5). Common to epigenetic mechanisms are

Table 2 Suggestions for future studies of genetic and genomic causes for severe asthma

Aim Tool
To evaluate the role of common Large-scale GWAS analyses of representative severe asthma
genetic variants cases (preferably >3000)
Targeted genotyping of SNPs in selected genes in large data
sets of severe asthma cases
To evaluate the role of structural Exon or whole-genome sequencing in a limited number of
and rare variants severe asthma cases followed by confirmatory analyses in larger data sets
To characterize the transcriptomic Tissue- and cell-specific global expression analyses of
profile of severe asthma samples collected from target organs (e.g. airway epithelial
and smooth muscle cells and peripheral blood cells)
To evaluate the role of epigenetic changes Global methylation analyses of samples from target organs
with regard to disease onset and progression (as above), collected at different time points (prior to disease,
at onset and when chronic disease is established)
To use functional studies and animal Functional characterization of key asthma genes may include
models for in-depth analyses of certain gene knockdown or knockout experiments (e.g. by siRNA shRNA
genes or pathways or gene deletion) and gene knockin experiments (e.g. by viral vectors)
To investigate whether patients with severe Geneenvironment interaction analyses using candidate genes as
asthma are particularly sensitive well as genome-wide data in combination with key exposure data
to certain exposures
To use a systems biology approach to Pathway analyses for example of gene expression data, or
understand the pathophysiology by combining information from genetic and genomic studies
of severe asthma

116 2012 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2012, 272; 108120
 E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma
effects on gene expression activity, resulting in in-
creased or decreased expression (exemplified by
ADAM33expressioninfibroblastsandepithelialcells,
as discussed previously). Epigenetic mechanisms
(such as DNA methylation) constitute a potential link
between geneticandenvironmentalfactors, as several
environmental factors have been shown to influence
methylation patterns [99]. Furthermore, studies have
shown that DNA methylation is altered in children ex-
posed to tobacco smoke or air pollutants (polycyclic
aromatic hydrocarbons) prenatally [100, 101]. So far,
epigenetic analyses within the respiratory field have
mostly been targeted to specific candidate genes such
as FOXP3 (involved in regulation of T-cell functions),
and no large-scale global methylation analyses on
asthma havebeenpresented[102, 103]. Thus, therole
of methylation status in childhood asthma remains
poorly understood, both at the single-gene level and at
theglobal genomiclevel.
Concluding remarks
Mapping the human genome has enabled genetic and
genomic research in relation to disease development
with unprecedented precision and speed. Although
the function of a large proportion of the human gen-
ome is still unknown, we are now beginning to under-
stand the role of certain genes and their protein prod-
ucts for human physiology and disease development.
The genetic research community has entered a new
era in which it is possible to map an individuals com-
plete DNA sequence and epigenetic profile, as well as
the complete transcriptomic and proteomic profiles.
In this review, we have discussed the recent findings
from genetic and genomic research studies of asthma,
focusing on severe asthma, and highlighted specific
genes for which there is much evidence for involve-
ment in asthma pathogenesis. It has become evident
that many genes contribute to asthma susceptibility,
with key roles suggested for genes involved in signal
transduction and immune processes. However, bio-
ontologic enrichment analyses of recently identified
asthma-related genes clearly show the limitations of
current bioinformatics tools, as several new asthma-
susceptibility genes such as ORMDL3 and IL33 were
not captured by the ontologic attributes and pathway
terms. Whilst much effort has been directed towards
understanding the genetic and genomic causes for
asthma in general, little is currently known about the
causes for severe, therapy-resistant asthma in chil-
dren. Suggestions for future studies to unravel the
geneticandgenomiccausesforsevere asthmaare out-
lined in Table 2. Because severe asthma is relatively
rare, large cohorts with this condition have been diffi-
cult to recruit, and therefore, collaboration between
research groups is needed. New insights into the path-
ogenesis of severe asthma are emerging, but further
investigation iswarranted.
Conflict of interest statement
No conflicts of interest to declare.
Acknowledgements
This work was supported by the Institute of Environ-
mental Medicine, Karolinska Institutet, the Swedish
Heart and Lung Foundation, the Swedish Founda-
tion for Strategic Research and the European Com-
missions Seventh Framework Programme under
grant agreement No. 261357 (MeDALL).
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Correspondence: Erik Melen, MD, PhD, Institute of Environmental
g 13 Box 210, SE-171 77
Medicine, Karolinska Institutet, Nobels va
Stockholm, Sweden.
(fax: +46-8-304571; e-mail: erik.melen@ki.se).