Discussion
This manuscript reports polygenic effects of multiple genetic variants
as well as modulating effects of environmental exposures on risk of
recurrent wheeze and asthma during early childhood. First, we determined
GRS for recurrent wheeze and asthma based on four genetic variants
located on chromosome 17q12-21. These loci were previously associated
with childhood-onset asthma in the largest published GWAS to date.(15)
In addition, we observed that breastfeeding until one year reduced
prevalence of recurrent wheeze, especially among children with moderate
or low genetic risk but not among those with high genetic risk. This
suggests that breastfeeding at one year of life reduces risk of
recurrent wheeze but does not offset high genetic risk. In contrast,
NO2 exposure appeared to increase the risk of
respiratory outcomes, particularly among children with moderate or high
genetic risk but did not affect those in the low GRS group. Overall, our
findings support the notion that asthma is a polygenic disease that is
modulated by interactions with environmental exposures, which could
account in part for the missing heritability. Moreover, our results
indicate that while asthma may be difficult to diagnose in
preschool-aged children, risk of respiratory outcomes can be detected
during early infancy using recurrent wheeze between ages 2-5 years.
Our GWAS of recurrent wheeze identified 98 associated SNVs, albeit all
are in strong linkage disequilibrium (LD, r2> 0.8), suggesting that they represent a single associated
locus. Of these, 19 have been previously correlated with asthma or a
related trait (e.g., allergy, atopic march, hay fever and eczema) and
only two are coding. For example, SNV rs2305479 is a missense variant
located within the GSDMB gene, resulting in an amino acid
substitution (Glycine to Arginine at position 304/416 of the Gasdermin-B
protein), and has been previously implicated in inflammatory processes.
An earlier study reported that rs2305479 leads to abnormal sulfatide
transport, which might compromise the integrity of the epithelial cell
barrier and promote inflammatory processes in inflammatory bowel disease
(IBD).(37) In addition, rs11078928 codes for a splice acceptor, which
could result in an alternative mRNA isoform. Further experiments are
needed to determine the biological effects of these genetic variants as
well as others in the same associated LD block.
In addition to SNVs associated with recurrent wheeze on chromosome 17,
which is a well-established locus for childhood asthma, we identified
that SNV rs145454327 on chromosome 1 was also associated with asthma by
age 5 years.(15) This intronic variant was previously associated with
childhood-onset asthma in the largest GWAS to date.(15) Furthermore,
chromatin state model analysis of fetal lung tissue identified that this
variant could potentially alter the motif for 4 transcription factors,
GATA-4, Tbp, FOXP1, and Sox8. GATA-4 have been previously implicated
with pathogenesis in bronchial asthma (38) and FOXP1 is known to
regulate epithelial cell fate and regeneration during lung
development.(39)
While our GWAS of recurrent wheeze and asthma both identified
associations with loci previously associated with asthma, there was no
overlap of associated loci. These results may reflect the phenotypic
heterogeneity of asthma as well as challenges in clinical diagnosis
among young children before age 7 (when spirometry is difficult and the
use of methacholine challenge tests are not typical).(16,17) In
addition, we combined definite and possible asthma into one case group
for GWAS, which may have further affected the accuracy of asthma
diagnosis by age 5. However, the association of SNPs on chromosome
17q12-21, a well-replicated locus for childhood-onset asthma, with
recurrent wheeze in this study indicates that asthma risk may be
assessed among young children, even prior to the diagnosis of asthma.
In addition to individual SNV associations with recurrent wheeze and
asthma, we identified the polygenic effects of four genetic variants
correlated with both recurrent wheeze (p = 1.53e-08) and asthma
diagnosis (p=9.39e-08). While the four variants identified in the GRS
analysis are non-coding, located either within intronic and 3’UTRs,
these have been previously identified to have potentially regulatory
effects. For example, the three risk variants associated with increased
prevalence of recurrent wheeze (rs3816470, rs8076131, and rs12603332)
are known loci that alter the expression of genes (eQTLs), as reported
by the Genotype-Tissue Expression (GTEx) project. Similarly, Tonchevaet al. reported that the protective SNV rs3902920 alters the
expression of ORMDL3 , a well-established asthma locus.(40) All
three risk variants identified in the GRS are correlated with increased
expression of ORMDL3/GSDMB in lung cells and immune cells
(leukocytes) while the protective variant is correlated with decreased
expression of the same genes. Studies have found that mice expressing
increased levels of human ORMDL3 or GSDMB have an asthma
phenotype characterized by increased airway responsiveness and increased
airway remodeling in the absence of airway inflammation.(41) This
suggests that the four variants used in calculating GRS in this study
may affect wheeze and asthma by regulating the expression of these
well-established asthma genes.
It is note-worthy that while GWAS of asthma did not yield associations
with loci on chromosome 17, our GRS analysis indicates that SNVs on this
chromosome may be used to assess risk of asthma as well as recurrent
wheeze. This may be explained by the modest effects of each individual
SNV on chromosome 17, which could not be detected in an univariate
analysis (i.e. GWAS) of single SNVs but the additive effects of these
variants could be detected in our GRS analysis.
In addition to polygenic effects of multiple genetic variants, we
determined that breastfeeding until 12 months and NO2exposure during the first 6 months of life significantly interact with
genetic factors to modulate risk of recurrent wheeze during early
childhood. Specifically, we observed lower prevalence of recurrent
wheeze among infants who were breastfed at 12 months compared to those
who were not breastfed until 12 months. The protective effects of
breastfeeding on wheeze in the CHILD study was previously reported by
Azad et al. (42) In the current study, this protective effect was
detected in children with modest or low genetic risk but not in the high
GRS group, which suggests that high genetic risk offsets the protective
effects of breastfeeding at one year of life. In contrast to
breastfeeding, we detected higher prevalence of recurrent wheeze among
infants exposed to higher NO2 in the first 6 months of
life. The detrimental effects on NO2 on recurrent
wheezing during the first two years of life in the CHILD study was
previously reported.(43) However, our study shows that this effect is
most evident among children with high genetic risk, whereby those
exposed to high NO2 have a 2.7-fold higher wheeze
prevalence than those exposed to low NO2.
This GRS study is the first to use results from the largest GWAS of
childhood-onset asthma published to date, which reported distinct
association signals between childhood vs. adult-onset disease,
suggesting different mechanisms of disease.(15) Prior GRS studies used
summary statistics from GWAS of both adult- and childhood-onset asthma.
Thus, our study uniquely focused on early-onset disease associations,
resulting in an associated GRS based on four genetic variations on
chromosome 17, which was the most significant peak from the
childhood-onset asthma GWAS. We observed more than a 2-fold higher
prevalence of recurrent wheeze and asthma between the high vs. low GRS
groups. Moreover, compared to single associated SNVs, the GRS accounts
for higher heritability estimates of recurrent wheeze and asthma (h2 =
2% vs. 1.4%). In fact, the GRS based on the additive effects of risk
alleles at these four loci accounts for a similar heritability as 31 of
the most replicated asthma variants (h2 = 2.5%). While a heritability
estimate of 2% may seem low, it was estimated that all common variants
account for up to 14% of asthma heritability.(5) Furthermore, we
demonstrated that genetic risk interacts with environment exposures,
which accounts for some of the missing heritability of asthma. In fact,
this interaction accounts for up to 3.6% of the heritability of
recurrent wheeze, which is an increase from considering the GRS alone.
While this manuscript reports a novel GRS of asthma and recurrent wheeze
that is based on additive effects of childhood-onset asthma genes and
identifies novel interactions with environmental exposures, our study
has several limitations. First, while our GRS analysis utilized results
from an independent GWAS of childhood asthma, the largest to date,
replication of our GRS results in an independent asthma cohort is needed
to validate our findings. A major challenge for replication is the
identification of another cohort with both genetics and environmental
exposures data in order to assess the effects of gene-environment
interactions on childhood asthma. A second limitation is that our
results are in silico -based and experimental studies are needed
to test the functional impact, if any, of the identified variants on
chromosomes 1 and 17. Given the large LD block on chromosome 17, which
consists of numerous SNVs spanning multiple genes, identification of
casual variants will be challenging. Finally, we have yet to assess
additional environmental exposures as well as the long-term effects
(i.e. beyond the first few year of life) on respiratory outcomes among
the children as they grow. For example, methacholine challenge tests are
ongoing in the CHILD Cohort Study at the age 8 clinical visits, which is
ongoing and will result in more accurate diagnosis of asthma.