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Click here for more articles from the symposium doi: 10.1111/j.1365-2796.2012.02555.x
Abstract. Melen E, Pershagen G (Institute of Environ- netic and genomic research studies of asthma, partic-
mental Medicine and Centre for Allergy Research, ularly severe asthma, and highlight specific genes for
Karolinska Institutet; Astrid Lindgren Childrens which there are multiple lines of evidence for involve-
Hospital, Karolinska University Hospital, Stock- ment in asthma pathogenesis. Bio-ontologic enrich-
holm, Sweden). Pathophysiology of asthma: lessons ment analyses of the most recently identified asthma-
from genetic research with particular focus on severe related genes point to attributes such as molecular
asthma (Reveiw). J Intern Med 2012; 272: 108120. and signal transducer activity and immune system
processes, which indicates the importance of immu-
There is good evidence that both inherited and envi- noregulation and inflammatory response in the path-
ronmental factors influence the risk of developing ogenesis of asthma. Finally, we discuss how genetic
asthma. Only recently, large well-designed studies and environmental factors jointly influence asthma
have been undertaken with the power to identify the susceptibility and summarize how the results may
genetic causes for asthma, and methods developed in increase understanding of the pathophysiology of
parallel with the Human Genome Project, such as asthma-related diseases.
gene expression and epigenetic studies, have made
large-scale analyses of functional genetics possible.
In this review, we discuss the recent findings from ge- Keywords: asthma, children,genetics, genomics, severity.
108 2012 The Association for the Publication of the Journal of Internal Medicine
E. Melen & G. Pershagen
| Review: Genetics and genomics of asthma
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- Data regarding DNA variants, gene expression and
MDL genes encode transmembrane proteins that are protein levels were all used in a study that lead to
anchored into the endoplasmic reticulum and are be- the identification of the CHI3L1 gene as a new asth-
lieved to be involved in protein folding. ORMDL genes ma-susceptibility locus [44]. The CHI3L1 gene en-
show high sequence conservation between many codes for YKL-40, a protein found in the lungs and
species, which supports a crucial role for cellular circulation that has been suggested to be a new bio-
functions. Specifically, it has been suggested that marker for severity of asthma [45]. Rather than
ORMDL3 is involved in cellular responses to inflam- using asthma as the phenotype, Ober et al. con-
mation and dysregulation of sphingolipid metabo- ducted a GWAS using serum YKL-40 levels as the
lism, but the exact mechanisms of action are still outcome. A promoter SNP (-131 C G) in CHI3L1
unclear [31]. was found to be strongly associated with elevated
YKL-40 levels (P = 1.1 10)13), as well as with asth-
The association between ORMDL3 variants and asth- ma and lung function. YKL-40, which is a chitinase-
ma has been confirmed in several large data sets [32 like protein, is produced by various inflammatory
35], including the largest GWAS of asthma to date cells and may have an important role in asthma
with more than 10 000 asthma cases and 16 000 development and disease deterioration. The associ-
controls (the GABRIEL study) [36]. A meta-analysis ations between CHI3L1 and asthma have been repli-
of five published studies of a particular SNP cated in other populations, but further studies on
(rs7216389) in nine populations demonstrated an asthma and asthma-related phenotypes are war-
odds ratio for asthma of 1.44 in individuals carrying ranted [24].
the risk allele [37]. This SNP was associated with
asthma and ORMDL3 expression with the highest de-
Recent asthma GWASs and bio-ontologic enrichment analysis
gree of statistical significance in the original GWAS
[29]. Association with increased asthma exacerba- As of January 2012, the results of 21 GWASs of
tions in children despite current medications has asthma or asthma-related traits had been published
also been reported [38], as well as with increasing and markers in 36 genes had been identified with a
asthma severity in adults [39]. However, the mecha- commonly used cut-off of P 10)6 for association
nisms underlying the increased risk of exacerbations (Table 1) [46]. To further explore the biology of the
are not fully known at present. Analyses stratified by genes identified in GWASs, we performed gene ontol-
age of onset suggest that ORMDL3 variants are partic- ogy (GO) enrichment analysis using the Database
ularly associated with childhood-onset asthma [40]; for Annotation, Visualization and Integrated Discov-
this finding was confirmed in the GABRIEL study ery (DAVID) [47, 48]. In terms of bio-ontologic
[36]. Clinical studies suggest that childhood and enrichment, the genes shown to be associated with
adult asthma differ with regard to a number of char- asthma in GWASs were enriched (P < 0.05) for onto-
acteristics, including sex preponderance, airflow logical attributes such as molecular signal trans-
obstruction, sensitization and structural changes of ducer activity (13 of the 36 identified genes: HLA-
the airways, although remodelling may already occur DQB1, IL18R1, IL2RB, IL1RL1, SMAD3, RORA, IL6R,
in children with severe asthma [4143]. HLA-DQA2, NOTCH4, GAB1, HLA-DPB1, HLA-DOA
and HLA-DRA) and immune system process (11 of
Although the genetic association between ORMDL3 the 36 genes: HLA-DQB1, IL18R1, IL1RL1, NOTCH4,
and childhood asthma seems very robust across SMAD3, IL13, IL6R, HLA-DPB1, HLA-DOA, HLA-
studies and populations, the disease risk association DQA2 and HLA-DRA). The enrichment results were
with a single genetic variant is modest as exemplified largely driven by two groups of genes represented by
by the above-mentioned meta-analysis. This is ex- the MCH class II genes and cytokine receptor activity
pected for a complex disease such as asthma, but lim- (IL18R1, IL2RB, IL1RL1 and IL6R), which indicates
its the use of single variants in genetic testing and the importance of immunoregulation and inflamma-
prediction algorithms. In addition, several variants in tory response in the pathogenesis of asthma. The
the chromosome 17q21 region, which also includes identified interleukin (IL) receptors, as well as the
the GSDML and ZPBP2 genes, are associated with IL33 loci confer involvement of the IL gene family
asthma but the mechanisms underlying the risk of and their receptors in asthma. IL33 with its receptor
asthma associated with these variants have not been IL1RL1 belongs to the IL1 family of cytokines that
fully elucidated. Gene expression studies have, how- are known to be expressed on epithelial cells and are
ever, given new insights into the genetic regulation of involved in the host response to certain environmen-
this region (see later for discussion). tal and infectious stimuli [49]. Severe asthma has
110 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
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 The suggested Kyoto Encyclopedia of Genes and Ge-
which indicated the importance of Th2-like genes in nomes (KEGG) pathways for this group of 36 asthma
the locus on chromosome 5q containing RAD50 and genes include asthma represented by the MCH class
IL13 [50], and TSLP on the same chromosome [36]. II genes and IL13 (Table 1). Thus, only six of the 36
In addition, association with HLA-DQB1 was ob- genes were captured by the KEGG terms, and the
served [50]. Using emergency department visits or same MCH genes were also captured by several other
hospitalizations as the definition of a severe asthma autoimmune diseases such as type I diabetes, auto-
exacerbation in the CAMP study, a combination of immune thyroid disease and systemic lupus erythe-
160320 SNPs were found to predict exacerbation matosus. This indicates that shared genetics is likely
with an area under the curve (AUC) of 0.66 [51]. For to be of importance for several diseases related to
a complex trait such as severe asthma, prediction inflammation and autoimmunity. It is well known
has proven to be very challenging, and the AUC in that the MCH genes are involved in these diseases,
this study shows that genetic analyses can improve and shared genetic effects have also been implicated
our diagnostic tools, although much work remains from previous studies on network analyses of com-
before clinical testing can become a reality. Amongst plex diseases [52]. However, most of the genes identi-
the top 160 SNPs identified in the CAMP study, one fied in GWASs to be associated with asthma were not
(rs10496476) is located within DPP10, which was captured by GO or KEGG pathway terms, suggesting
identified as an asthma-susceptibility gene by posi- that our current understanding of the role of these
tional cloning [14]. 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- the transcript sequence and ultimately the protein
sidered to be the strongest locus for childhood asth- product (Fig. 2). However, there are a number of regu-
ma, were not captured by the enrichment analysis. latory mechanisms in cells that will influence the
This highlights the limitations of the use of current transcription rate (e.g. binding of transcription fac-
databases and bioinformatics tools because informa- tors to the promoter region and epigenetic changes),
tion regarding new genes with unknown or only transcript sequence (posttranscriptional splicing or
partly known functions is inconsistent with previous alternative polyadenylation) and translation into pro-
knowledge of human physiology and disease patho- tein [60]. Regulatory, nonprotein-coding RNAs have
genesis. This does not mean that the encoded gene attracted much attention recently and are believed to
products and pathways implied by these new genes have important roles in transcriptional and posttran-
are not relevant for asthma pathogenesis. On the con- scriptional regulation [61]. In addition, environmen-
trary, it suggests that substantial efforts are needed tal stimuli may be potent triggers of expression of spe-
to clarify the role of these new genes for asthma and cific genes. Thus, determining the DNA sequence of a
related diseases. A review and detailed description of particular region will only provide the underlying ba-
GWAS-identified asthma-associated genes reported sis for biological functions that may be responsible
until 2010 and their involvement in asthma patho- for disease development and not the whole picture.
genesis was recently published [31]. By studying gene expression and protein characteris-
tics, we will obtain better functional information
about processes related to health and disease. Gene
Whole-genome sequencing
expression is in many cases tissue specific, which is
By design, rare variants or mutations are not cap- important to consider when studies are compared.
tured by current GWAS chips and thus cannot be On the other hand, analyses of tissues directly af-
evaluated in these studies. It has been suggested that fected by the disease (e.g. airway epithelial or smooth
rare variants are important for several complex dis- muscle cells from patients with asthma) may give
eases [53], but to date, few studies have addressed valuable insights into the pathophysiology of the tar-
the role of rare variants in the pathogenesis of asth- get organ.
ma. However, studies of, for example, FLG, IL4,
IL12RB1 and TACI point to a potentially important
Examples from candidate expression studies
role for rare variants in asthma pathogenesis [5457].
Similar to candidate gene studies of asthma, several
There is currently a very rapid development in candidate expression studies of asthma have also
sequencing technologies, usually referred to as next- been published during the last 15 years. Targeted
generation sequencing (NGS), in terms of accuracy, PCR primers for a specific region or gene are usually
genetic cover, speed and costs [58]. A major advan- designed, and the region of interest is amplified and
tage of whole-genome sequencing is the possibility of the amount of RNA quantified. For example, chronic
detecting genetic variants other than SNPs, including severe asthma has been shown to be accompanied by
rare variants, insertions deletions, inversions and
copy number variations (CNVs). NGS will in the near
future offer large-scale sequencing capabilities in ge- Transcription
netic studies as well as clinical settings. The 1000 Ge- DNA (antisense) 3T A A T T T G A T5
nomes Project (http://www.1000genomes.org/) is mRNA (sense) 5A U U A A A
the first project to sequence the genomes of a large
Codon
number of people with the aim of providing a compre-
hensive map of human genetic variation [59]. Translation
Amino acid Protein
112 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
an upregulation of TNF expression in peripheral port the findings of genetic associations. For exam-
blood cells and high levels of tumour necrosis factor ple, expression of GPRA NPSR1 was found to be
alpha (TNF-a) in bronchoalveolar lavage (BAL) fluid markedly increased in bronchial biopsies from pa-
[62, 63]. At the gene level, the TNF promoter G-308A tients with asthma compared with healthy controls,
in particular has been associated with enhanced in vi- and Gpra Npsr1 mRNA was significantly upregulated
tro transcription and increased TNF-a levels in white in lung tissue from sensitized mice compared with
blood cells [64]. From a recent meta-analysis, it was control mice [17]. Neuropeptide S (NPS) activates sig-
concluded that the -308A allele confers a significant nalling through its receptor NPSR1; the effect of
risk of asthma, which links TNF with asthma at the receptor activation on gene expression has been
DNA, RNA and protein levels [65]. characterized in cell lines using microarray analysis
[70]. This revealed a set of 300 genes with altered
One of the most studied candidate genes, IL13, for expression. One of the upregulated genes, showing a
which association with asthma and allergy has been robust >4-fold change, was Tenascin C (TNC). Subse-
well replicated, has also been the focus of substantial quently, it was shown that experimental NPS stimu-
functional studies, especially with regard to its broad lation of NPSR1-transfected cells upregulated TNC
involvement in allergic inflammation [66]. For exam- mRNA in a dose-dependent manner [71]. Epistatic ef-
ple, it has been proven that the IL13 promoter SNP- fects between NPSR1 and TNC variants also altered
1112CT has functional effects in that enhanced pro- the risk of allergic disease, and this interaction high-
moter activity and increased IL13 transcription are lights the complex interplay between several genes
seen in human and mouse Th2 cells [67]. The same and their products for the development of asthma
SNP has also been associated with both asthma and and allergy. For another positionally cloned gene,
allergy [22], and IL13 (IL4 IL13 locus) was the only ADAM33, increased protein levels in BAL fluid from
gene that showed associations with both asthma and patients with severe asthma supports its role in dis-
an allergy-related trait (total IgE) in a GWAS [36]. Thy- ease development [72]. Expression of ADAM33 in epi-
mic stromal lymphopoietin (TSLP) is a cytokine re- thelial and fibroblast cells from bronchial biopsy
leased from airway epithelial cells in response to specimens has been shown to be controlled by epige-
pathogens or inflammatory cytokines such as IL13. netic mechanisms (methylation status of a regulatory
Two studies support its involvement in the pathogen- CpG island within the ADAM33 promoter) [73]. Of
esis of severe asthma: a functional study showed that importance, these results demonstrate that (i) gene
TSLP protein expression was significantly increased expression can be very cell specific even in adjacent
in airway epithelium of patients with asthma, partic- cell tissues both relevant for a certain disease (e.g.
ularly those with severe disease [68], and results of expression of ADAM33 in fibroblasts but not epithe-
the GABRIEL study suggested an association be- lial cells in asthma) and (ii) expression can be tightly
tween TSLP SNPs and severe asthma [36]. controlled by epigenetic mechanisms as low levels of
methylation were detected in fibroblasts that express
IL33 is another example of a member of the IL family ADAM33, whereas epithelial cells that do not express
for which evidence of involvement in asthma patho- ADAM33 showed a very high level of promoter methyl-
genesis has been provided from various sources in re- ation (see further discussion of epigenetics later).
cent years [49]. In addition to being identified in re-
cent GWASs, functional studies suggest that IL33
Whole-genome expression
may have a key role in host responses to stimuli such
as exposure to allergens, air pollutants and respira- The transition from a candidate gene to a whole-gen-
tory viruses that may cause damage to the airway ome approach for DNA variants has been accompa-
through IL33 activation and inflammation triggering. nied, or even preceded by, similar development in
IL33 may also catalyse sensitization to allergens expression analysis. The method of whole-genome
through dendritic cell activation and Th2-mediated expression analysis has been available for more than
pathology. Furthermore, IL33 is upregulated in air- a decade, enabling an unbiased view of upregulated
way smooth muscle cells, which are key cells in the and downregulated genes in relation to a specific phe-
regulation of bronchial responsiveness and airway notype or disease. However, there have been rela-
tonus, from patients with severe asthma, suggesting tively few studies of human asthmatic tissue samples
an involvement in therapy-resistant asthma [69]. and most have been hampered by small sample sizes.
Using a global approach, hundreds or thousands
In several positional cloning studies related to asth- of genes may be differentially expressed between
ma, gene expression patterns have been used to sup- 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- asthma. Until recently, theTAS2Rs were not known
cated to interpret at an individual gene level, and net- to have any clear role in asthma. However, it has been
work or pathway approaches are therefore attractive found that the TAS2Rs are expressed in human air-
options. This field is rapidly evolving under the um- way smooth muscle and that activation of these
brella term systems biology [74]. Within an ongoing receptors causes relaxation of smooth muscles and
large European collaborative project (MeDALL), ef- dilatation of airways. Inhaled bitter tastants also
forts are being made to bridge the gap between func- caused decreased airway obstruction in a mouse
tional genetic research and systems biology with clin- model of asthma [80]. The bronchodilating effects of
ical and epidemiological research to increase our TAS2R agonists have been replicated in other airway
understanding of the causes for asthma and allergic models [81]. Based on these findings, the expression
disease [75]. of TAS2Rs in peripheral blood cells in patients with
severe asthma is currently under further examina-
Severe asthma exacerbations in children are often tion to clarify the role of these receptors in asthma.
triggered by viral infections, but the cellular and
molecular mechanisms associated with these exacer- Figure 3 shows a simplified schematic diagram of our
bations are poorly understood. Using a genomics- current understanding of the genetic and genomic
based approach involving microarray profiling of contributions to severe asthma, where association at
peripheral blood mononuclear cells collected during the DNA or RNA level has been established. For some
an acute exacerbation and afterwards during conva- of the associated genes, there is also evidence of
lescence, gene expression patterns were studied in a altered protein secretion in cells with a key role in
cohort of asthmatic children [76]. The results of a sim- asthma, as indicated in Fig. 3.
ilar study in adults have also been published recently
[77]. The genes identified as differentially expressed Moffatt et al. [29] have provided an illustrative exam-
during exacerbation and convalescence in children ple of a successful combination of association and
comprised genes associated with (i) arachidonic gene expression analyses for ORMDL3. In addition to
acid prostaglandin metabolism, (ii) leucocyte migra- the GWAS analysis showing strong association to the
tion, (iii) innate immunity, (iv) adaptive immunity, chromosome 17q21 region, global gene expression in
(v) the complement coagulation cascade, and (vi) Epstein-Barr virus-transformed lymphocytes (EB-
inflammation. One of the most highly upregulated VLs) from 378 asthmatic children and their siblings
genes during acute exacerbation was CCR2, previ- was measured. The same subjects had been geno-
ously shown to play an important role in trafficking of typed with a GWAS platform (Illumina). EBVLs repre-
dendritic cells and monocytes to the lung during air- sent the B-cell lineage and have been shown to have
way inflammation. A key finding was the upregula- direct relevance to asthma. Transcripts in one gene,
tion of another well-known gene involved in allergic ORMDL3, were strongly (P < 10)22) associated with
responses and regulation of IgE levels, FceR1 (the exactly the same SNPs as found in the GWAS. It was
high-affinity IgE receptor), which suggests that respi- further estimated that the disease-associated SNPs
ratory viral infections in children may initiate an ato- (also known as expression quantitative trait loci, eQ-
py-dependent cascade of signalling events. FceR1 has TLs, when expression is affected) accounted for
also been significantly associated with total IgE levels 29.5% of the variance of ORMDL3 expression.
in peripheral blood in two GWASs [36, 78].
The fact that several markers at the chromosome
Using genome-wide expression arrays, the tran- 17q21 locus were associated with asthma in the origi-
scriptomes of asthmatic children from Sweden were nal and follow-up studies, along with the strong link-
characterized, and a difference in gene expression be- age disequilibrium between these markers, makes it
tween severe, therapy-resistant asthma and mild impossible to identify one specific variant that is more
asthma could be observed [79]. Traditional immune- important than any others as the definitive risk vari-
related functions, such as T-cell receptor signalling ant. In addition, ORMDL3 has been associated with
and natural killer cell-mediated cytotoxicity, were in autoimmune diseases such as Crohns disease and
this study found to be upregulated in children with type 1 diabetes, which suggests that this locus may
controlled asthma, but such patterns were not ob- harbour variants that exert transcriptional control
served in those with severe asthma. Instead, a signifi- with broad effects [82, 83]. Verlaan et al. [84] under-
cant enrichment and upregulation of bitter taste took a series of in-depth functional experiments in
receptors (TAS2Rs) was found, which highlighted lymphoblastoid cell lines and demonstrated that the
new pathways contributing to therapy-resistant genetic variants associated with asthma act over a
114 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
Airway
Protein sectretion:
TNF-
Epithelial cells TSLP
ADAM33
IL33
Transcription
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
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
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- rare, large cohorts with this condition have been diffi-
creased or decreased expression (exemplified by cult to recruit, and therefore, collaboration between
ADAM33expressioninfibroblastsandepithelialcells, research groups is needed. New insights into the path-
as discussed previously). Epigenetic mechanisms ogenesis of severe asthma are emerging, but further
(such as DNA methylation) constitute a potential link investigation iswarranted.
between geneticandenvironmentalfactors, as several
environmental factors have been shown to influence
Conflict of interest statement
methylation patterns [99]. Furthermore, studies have
shown that DNA methylation is altered in children ex- No conflicts of interest to declare.
posed to tobacco smoke or air pollutants (polycyclic
aromatic hydrocarbons) prenatally [100, 101]. So far,
Acknowledgements
epigenetic analyses within the respiratory field have
mostly been targeted to specific candidate genes such This work was supported by the Institute of Environ-
as FOXP3 (involved in regulation of T-cell functions), mental Medicine, Karolinska Institutet, the Swedish
and no large-scale global methylation analyses on Heart and Lung Foundation, the Swedish Founda-
asthma havebeenpresented[102, 103]. Thus, therole tion for Strategic Research and the European Com-
of methylation status in childhood asthma remains missions Seventh Framework Programme under
poorly understood, both at the single-gene level and at grant agreement No. 261357 (MeDALL).
theglobal genomiclevel.
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Medicine, Karolinska Institutet, Nobels va
6907. Stockholm, Sweden.
98 Ng SF, Lin RC, Laybutt DR, Barres R, Owens JA, Morris MJ. (fax: +46-8-304571; e-mail: erik.melen@ki.se).
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