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Environmental and occupational disease
Early-life inhalant allergen exposure, filaggrin
genotype, and the development of sensitization
from infancy to adolescence
Angela Simpson, MD, PhD,
a
* Helen A. Brough, MD, PhD,
b,c,d
* Sadia Haider, PhD,
e,f
* Danielle Belgrave, PhD,
g
Clare S. Murray, MD,
a
and Adnan Custovic, MD, PhD, FAAAI
e,f
*Manchester, London, and Cambridge, United Kingdom
GRAPHICAL ABSTRACT
Early-life inhalant allergen exposure, filaggrin genotype and the development of sensitization from infancy to
adolescence
FLG loss-of-funcon vs wild-type
Feld d 1
Der
p
1
Can f 1
Exposure in the
first year of life
Longitudinal risk of specific sensizaon to cat, HDM and dog, and any sensizaon to any
allergen from infancy to adolescence
OR 1.36 (95% CI 1.02-1.80); p=0.035
OR 1.09 (95% CI 0.78-1.51); p=0.62
OR 0.76 (95% CI 0.47-1.22); p=0.26
OR 0.16 (95% CI 0.03-0.86); p=0.032
OR 2.89 (95% CI 0.69-12.04); p=0.145
From
a
the Division of Infection, Immunity and Respiratory Medicine, Faculty of
Biology, Medicine and Health, Manchester Academic Health Sciences Centre, Univer-
sity of Manchester and University Hospital of South Manchester NHS Foundation
Trust;
b
the Children’s Allergy Service, Evelina London, Guys and St Thomas’ NHS
Trust, London;
c
the Paediatric Allergy Group, Department of Women and Children’s
Heath, School of Life Course Sciences, London;
d
the Paediatric Allergy Group, School
of Immunology & Microbial Sciences, King’s College London;
e
the Section of Paedi-
atrics and
f
the National Heart and Lung Institute, Imperial College London; and
g
Mi-
crosoft Research Cambridge.
*These authors contributed equally to this work as joint first authors.
Supported by UK Medical Research Council grants MR/K002449/1 and MR/LO12693/
1. The Manchester Asthma and Allergy Study was supported by Asthma UK grants
301 (1995-1998), 362 (1998-2001), 01/012 (2001-2004), and 04/014 (2004-2007);
the BMA James Trust (2005) and the JP Moulton Charitable Foundation (2004-
current); the North West Lung Centre Charity (1997-current); and Medical Research
Council grants G0601361 (2007-2012), MR/K002449/1 (2013-2014), and MR/
L012693/1 (2014-2018). This report is independent research supported by the National
Institute for Health Research (NIHR) South Manchester Respiratory and Allergy Clin-
ical Research Facility at Manchester University NHS Foundation Trust (Wythen-
shawe) and by the NIHR Manchester Biomedical Research Centre. The views
expressed in this publication are those of the authors and not necessarily those of
the National Health Service, the NIHR, or the Department of Health. A.S. is supported
by the Manchester Biomedical Research Centre.
Disclosure of potential conflict of interest: A. Simpson reports lecture fees from Thermo
Fisher Scientific. H. A. Brough reports advisory board work for DBVand research sup-
port from Thermo Fisher Scientific outside of the submitted work. A. Custovic reports
personal fees from Novartis, Regeneron/Sanofi, Thermo Fisher Scientific, Boehringer
Ingelheim, Novartis, and Philips outside the submitted work. The rest of the authors
declare that they have no relevant conflicts of interest.
Received for publication November 11, 2018; revised July 18, 2019; accepted for publi-
cation August 21, 2019.
Available online October 17, 2019.
Corresponding author: Adnan Custovic, MD, PhD, FAAAI, Imperial College London, St
Mary’s Campus Medical School, London W2 1PG, United Kingdom. E-mail:
a.custovic@imperial.ac.uk.
The CrossMark symbol notifies online readers when updates have been made to the
article such as errata or minor corrections
0091-6749
Ó2019 The Authors. Published by Elsevier Inc. on behalf of the American Academy of
Allergy, Asthma & Immunology. This is an open access article under the CC BY li-
cense (http://creativecommons.org/licenses/by/4.0/).
https://doi.org/10.1016/j.jaci.2019.08.041
993
Background: We hypothesized that filaggrin (FLG) loss-of-
function mutations modify the effect of allergen exposure on the
development of allergic sensitization.
Objective: We sought to determine whether early-life exposure
to inhalant allergens increases the risk of specific sensitization
and whether FLG mutations modulate these odds.
Methods: In a population-based birth cohort we measured mite,
cat, and dog allergen levels in dust samples collected from
homes within the first year of life. Sensitization was assessed at 6
time points between infancy and age 16 years. Genotyping was
performed for 6 FLG mutations.
Results: In the longitudinal multivariable model (age 1-16
years), we observed a significant interaction between FLG and
Fel d 1 exposure on cat sensitization, with the effect of exposure
being significantly greater among children with FLG mutations
compared with those without (odds ratio, 1.36; 95% CI, 1.02-
1.80; P5.035). The increase in risk of mite sensitization with
increasing Der p 1 exposure was consistently greater among
children with FLG mutations, but the interaction did not reach
statistical significance. Different associations were observed for
dogs: there was a significant interaction between FLG and dog
ownership, but the risk of sensitization to any allergen was
significantly lower among children with FLG mutations who
were exposed to a dog in infancy (odds ratio, 0.16; 95% CI,
0.03-0.86; P5.03).
Conclusions: FLG loss-of-function mutations modify the
relationship between allergen exposure and sensitization, but
effects differ at different ages and between different allergens.
(J Allergy Clin Immunol 2020;145:993-1001.)
Key words: Allergen exposure, house dust mite, cat, dog, sensitiza-
tion, birth cohort, filaggrin, childhood, Fel d 1, Der p 1, Can f 1
Although being exposed to an allergen or allergens is a
prerequisite for the development of sensitization, the nature of
the relationship between the level of exposure and the risk of
sensitization is unclear.
1,2
For example, in some studies exposure
to house dust mite (HDM) allergens has been shown to increase
the risk of HDM sensitization and asthma,
3-6
particularly in chil-
dren with parental atopy.
7,8
However, other studies have not
confirmed this association (reviewed by Custovic
1
).
9
Similarly,
conflicting data have been reported on the effect of cat ownership
and Fel d 1 exposure on cat sensitization, which have been shown
in different studies to be either risk
6,9-11
or protective
12,13
factors.
Early-life exposure to dogs in the home has been shown to reduce
subsequent risk of allergic sensitization to multiple allergens,
14
but no studies have assessed the effect of objectively measured
dog allergen levels in homes on specific sensitization.
A recent study has reported different associations between
early-life cat exposure and sensitization to cat at different ages in
a birth cohort, pointing to the importance of life-course perspec-
tive.
15
In the first 3 years of life, sensitization was more common
among cat owners, but after this, the increase in sensitization rate
was greater among children without a cat, and therefore by
adolescence, the prevalence of sensitization was numerically
greater in this group (although the difference was not statistically
significant).
15
Hence apparently contradictory results might be a
consequence of different life-course sensitization trajectories be-
tween exposed and nonexposed subjects. Therefore, to under-
stand the complex relationship between early-life exposures and
later clinical outcomes, one should not rely only on cross-
sectional analyses because more useful information can be gained
through analysis of longitudinal trajectories.
1,16
The effect of early allergen exposure on sensitization is
modified by parental atopy and birth order,
17
alluding to the
importance of both genetic and environmental factors.
1
The
concept that the same environmental exposure can have different
effects among subjects with different genetic predisposition has
been tested in studies that assessed the interaction between genes
and the susceptibility to environmental factors.
18-20
Variability in
response to HDM exposure in relation to mite-specific sensitiza-
tion has been attributed to the IL4 gene promoter polymorphism
C-590T.
21
Filaggrin (FLG) loss-of-function mutations contribute
to an impaired skin barrier and are associated with eczema and a
range of allergic conditions,
22-24
as well as allergic sensitiza-
tion.
24,25
Children with FLG mutations were found to have an
increased risk of eczema if they were exposed to cat in early
life, with no effect of exposure among those without FLG
mutations.
26
In a study on food allergy, we have shown that early-life
exposure to peanut allergens measured in dust collected from
homes is associated with an increased risk of peanut sensitization
and allergy in children who carry FLG mutations, with no
significant effect of exposure in those without FLG mutations.
27
In the current study we hypothesized that FLG loss-of-function
mutations would modify the effect of exposure to inhalant
allergen (HDM, cat, and dog) on the development of
sensitization. To test this, we used both cross-sectional and
longitudinal analyses to investigate the effect of early-life
domestic allergen exposure on subsequent sensitization and
whether these relationships were altered by FLG genotype and
modified over time.
METHODS
Study design, setting, participants, data sources,
and definition of outcomes
The Manchester Asthma and Allergy Study is an unselected birth cohort
described in detail elsewhere.
28
A detailed description is provided in the
Methods section in this article’s Online Repository at www.jacionline.org.
Briefly, 1184 subjects were recruited prenatally and followed prospectively.
For this study, we used data from 1051 children in the observational cohort,
excluding 133 children who took part in the environmental intervention
arm.
29,30
The study was approved by the local ethics committee, and parents
provided written informed consent.
Participants attended follow-up visits at ages 1, 3, 5, 8, 11, and 16 years. We
assessed sensitization by skin prick tests. Mite, cat, and dog sensitization were
defined as a wheal diameter at least 3 mm larger than that elicited by the
negative control. Allergic sensitization was defined as a positive skin prick test
response to at least 1 of the allergens tested. Cat and dog ownership in the first
year of life was ascertained by using questionnaires administered at home visit
in the first year of life.
Abbreviations used
AD: Atopic dermatitis
FLG: Filaggrin
GEE: Generalized estimating equations
HDM: House dust mite
OR: Odds ratio
J ALLERGY CLIN IMMUNOL
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994 SIMPSON ET AL
Quantification of allergen exposure
Dust samples were collected in the first year of life from the living room and
child’s bedroom floors.
31
Der p 1, Fel d 1, and Can f 1 levels were measured by
using an mAb-based ELISA (Indoor Biotechnologies, Cardiff, United
Kingdom) with a detection limit of 0.2 mg/g, as previously described.
32,33
To determine an individual child’s allergen exposure, we averaged
allergen concentrations in samples taken from the living room and child’s
bedroom.
Genotyping
FLG genotyping was performed by using probes and primers, as previously
described.
26,27,34
Genotyping for R501X, S3247X, and R2447X mutations
was performed with a TaqMan-based allelic discrimination assay (Applied
Biosystems, Cheshire, United Kingdom). Mutation 2282del4 was genotyped
by sizing of a fluorescently labeled PCR fragment on a 3100 or 3730 DNA
sequencer. FLG mutations 3673delC and 3702delG were assessed by using
GeneScan analysis of fluorescently labeled PCR products. Data were analyzed
as combined carriage of an FLG-null allele; that is, children carrying 1 or more
of the 6 genetic variations were considered to have an FLG loss-of-function
mutation.
27
In cases with incomplete FLG data, the presence of 1 FLG muta-
tion defined that case as a carrier; participants with incomplete genotyping
data in whom all alleles successfully tested were wild-type were excluded
from further analysis because it was not possible to determine their FLG ge-
notype status.
27
Statistical analysis
Allergen levels (expressed in micrograms per gram) underwent natural log
transformation. The effects of FLG loss-of-function mutations and allergen
exposure on allergen-specific sensitization at each age were investigated by
using logistic regression. We first analyzed the associations between sensitiza-
tion and allergen exposure in each FLG genotype group.
We then modeled the effect of the interaction between exposure and FLG,
controlling for the main effects. Longitudinal analyses were performed by us-
ing generalized estimating equations (GEE). Population-averaged GEE
models were developed to investigate whether the effect of allergen exposure,
FLG loss-of-function mutation, and their interactions on the development of
sensitization changed over time. Coefficients represent the increased/
decreased odds of sensitization per log-unit increase in allergen exposure.
We also investigated the effect of cat and dog ownership in early life on
sensitization. We adjusted all models with confounding variables, including
sex, socioeconomic status, and breast-feeding.
We tested the assumption of a linear relationship between allergen
exposure and sensitization by conducting likelihood ratio tests to compare
the fit of nested models with inclusion and exclusion of a quadratic term for
each exposure at all time points. Furthermore, link tests were carried out to
check for model misspecification of the dependent variable when only a linear
term for exposure was included.
35
Given a smaller sample size at age 1 year, we assessed the sensitivity of our
findings from longitudinal analyses with the exclusion of data at age 1 year. All
analyses were conducted in Stata 15 software (StataCorp, La Jolla, Calif).
RESULTS
The flow of children through the study is summarized in Fig E1
in this article’s Online Repository at www.jacionline.org. Com-
plete FLG genotyping was available for 712 (76.3%) of 933 white
participants, of whom 131 did not have dust samples. We
analyzed data from 581 children, of whom 51 (8.8%) had FLG
loss-of-function mutations; 276 had complete sensitization data
from age 3 to 16 years. Excluded participants were more likely
to be male and have paternal asthma, but there were no differences
in other risk factors and exposures, including FLG genotype and
pet ownership (see Table E1 in this article’s Online Repository at
www.jacionline.org).
Table E2 in this article’s Online Repository at www.jacionline.
org shows the prevalence of sensitization from age 1 to 16 years in
the whole population and stratified by FLG genotype. For all al-
lergens, children with FLG mutations had significantly greater
point prevalence of sensitization in preschool (age 1-5 years)
and for cat and HDM by mid–school age (age 11 years), but there
were no differences in sensitization between genotype groups in
adolescence (age 16 years, see Fig E2 in this article’s Online Re-
pository at www.jacionline.org).
Early-life allergen exposure, FLG genotype, and
allergen-specific sensitization
Cat. Cross-sectional analyses in each FLG genotype group
suggested that the effect of early-life Fel d 1 exposure on cat
sensitization differed between children with and without FLG
mutations (Fig 1). Among children with mutations, an increase
in exposure significantly increased the risk of sensitization at
ages 1, 3, 5, and 8 years; this association was no longer significant
thereafter (see Table E3 in this article’s Online Repository at
www.jacionline.org). Among children with a wild-type genotype,
there was a significant association between Fel d 1 exposure and
cat sensitization at age 1 year, with no significant association at
later ages. The effect of early-life exposure on sensitization
diminished over time in both genotype groups. In longitudinal
adjusted GEE models, Fel d 1 exposure significantly increased
the risk of sensitization among children with FLG mutations
(odds ratio [OR], 1.26; 95% CI, 1.04-1.52; P5.017), with no
significant effect of exposure in those without mutations (OR,
0.94; 95% CI, 0.84-1.06; P5.34). Similar results were obtained
in 230 children with complete data from age 3 to 16 years (see
Table E3).
These observations suggesting an interaction between FLG
genotype and Fel d 1 exposure were formally tested in models
that included an interaction term controlling for main effects
(Table I). The effect of early-life exposure on cat sensitization
was significantly greater in the FLG mutation group at the ages
of 5 years (OR, 1.99; 95% CI, 1.05-3.79; P5.035) and 8 years
(OR, 1.59; 95% CI, 1.07-2.37; P5.02). In the longitudinal
GEE model we observed a significant interaction between FLG
genotype and Fel d 1 exposure in that the effect of early-life
exposure on development of cat sensitization from infancy to
age 16 years was significantly greater among children with
FLG mutations compared with those without (OR, 1.36; 95%
CI, 1.02-1.80; P5.035; Table I). The interaction effect remained
robust to sensitivity testing among participants with complete
data.
HDM. Analyses in each FLG genotype group suggested a
broad pattern similar to that observed for cat allergen (Fig 2).
At age 1 year, the effect of Der p 1 exposurewas markedly greater
in children with FLG mutations (OR, 6.66; 95% CI, 1.15-38.58;
P5.03); from age 3 years onward, ORs for the effect of Der p
1 exposure were numerically higher among children with FLG
mutations, but this did not reach statistical significance (see
Table E3). In longitudinal models we observed nonsignificant
trends for the increase in risk of sensitization with increasing
Der p 1 exposure in both genotype groups (OR, 1.11 [95% CI,
0.99-1.25; P5.06] and 1.31 [95% CI, 0.96-1.80; P5.09],
wild-type and FLG mutations, respectively; see Table E3).
Although the increase in risk per increase in unit of Der p 1 expo-
sure was consistently greater among children with FLG
J ALLERGY CLIN IMMUNOL
VOLUME 145, NUMBER 3
SIMPSON ET AL 995
mutations, except at age 16 years (see Table E3), in multivariable
models the interaction between FLG and exposure did not reach
statistical significance (Table I).
Dog. The relationship between Can f 1 exposure and dog
sensitization differed from that observed for cat and HDM (Fig 3).
Analyses in each genotype group showed that Can f 1 exposure in
children without FLG mutations increased the risk of
sensitization, with the effect being significant at age 16 years
(OR, 1.26; 95% CI, 1.06-1.50; P5.001) and no significant effect
of exposure among children with FLG mutations (see Table E3).
In GEE models the effect of Can f 1 exposure differed between
genotype groups, with a significant increase in the risk of
sensitization among children without FLG mutations (OR, 1.20;
95% CI, 1.06-1.37; P5.004) but not among those with FLG
mutations. The formal interaction analyses showed that the effect
of Can f 1 exposure on dog sensitization was consistently lower at
all ages in children with FLG mutations, but the interaction
between FLG and dog allergen exposure did not reach
significance (Table I).
For all allergens and models, a quadratic term did not improve
the explanatory power of the relationship between exposure and
sensitization (results are available on request).
Pet ownership in the first year of life, FLG genotype,
and sensitization during childhood
Fig E3,A, in this article’s Online Repository at www.
jacionline.org shows the proportions of cat-sensitized children
by FLG genotype and cat ownership in the first year of life.
From infancy to age 11 years, children with FLG mutations and
a cat at home had the greatest risk of sensitization; the probability
of sensitization converged to a similar level by age 16 years in all
4 groups (see Fig E4 in this article’s Online Repository at www.
jacionline.org). Adjusted cross-sectional analyses (see Table E4
in this article’s Online Repository at www.jacionline.org) consis-
tently showed that from age 3 to 8 years, children with FLG mu-
tations and a cat at home had the greatest risk of cat sensitization
(approximately 4-fold greater risk compared with those without
mutations and no cat). Longitudinal analyses demonstrated that
children with FLG mutations and a cat at home had the greatest
probability of cat sensitization during childhood, which was
significantly greater compared with children with no cat and
wild-type FLG (OR, 3.02; 95% CI, 1.26-7.21; P5.013; see
Table E4). The results of the adjusted longitudinal model are
presented in Table E5 in this article’s Online Repository at
www.jacionline.org.
FIG 1. Effect of exposure to cat allergen Fel d 1 on the predicted probability of cat-specific sensitization
among children with and without FLG loss-of-function mutations: ages 1, 3, 5, 8, 11, and 16 years. Ln, Nat-
ural log.
J ALLERGY CLIN IMMUNOL
MARCH 2020
996 SIMPSON ET AL
These relationships were different for dog ownership. Children
with FLG mutations and a dog at home had the lowest point prev-
alence of dog sensitization at all ages apart from 5 years (see Fig
E3,B). In this group there were no dog-sensitized subjects at 5
follow-ups, rendering the results of cross-sectional analyses un-
certain (see Table E6 in this article’s Online Repository at
www.jacionline.org). Models indicated that dog ownership
among children with FLG mutations was protective (see Fig E5
in this article’s Online Repository at www.jacionline.org), but
formal statistical significance of the interaction in GEE models
was not achieved (OR, 0.06; 95% CI, 0.00-1.72; P5.10; see
Table E7 in this article’s Online Repository at www.jacionline.
org).
To investigate whether dog ownership can offer protection
that is not allergen specific, we proceeded to analyze sensiti-
zation to any allergen. Fig E6 in this article’s Online Reposi-
tory at www.jacionline.org shows the proportions of
sensitized children according to FLG genotype and dog owner-
ship. A consistent finding was decreased risk of sensitization
among dog owners with FLG mutations, with the opposite ef-
fect in those without mutations (Fig 4). In the longitudinal GEE
model there was a significant interaction between FLG geno-
type and dog ownership in that the risk of sensitization was
markedly and significantly lower among children with FLG
mutations who had a dog in the home in infancy (OR, 0.16;
95% CI, 0.03-0.86; P5.03; Table II). There was no effect
of cat ownership on sensitization to any allergen and no inter-
action with FLG (see Table E8 in this article’s Online Repos-
itory at www.jacionline.org).
DISCUSSION
In this population-based birth cohort children with FLG muta-
tions were more likely to be sensitized to inhalant allergens from
infancy to school age, but there were no differences in sensitiza-
tion between those with and without FLG mutations in adoles-
cence. Longitudinal sensitization trajectories differed between
children exposed to an allergen or allergens in the first year of
life compared with those not exposed and between genotype
groups. In general, the effect of cat and mite allergen exposure
on allergen-specific sensitization was greater among children
with FLG loss-of-function mutations compared with those
without. We have shown a significant interaction between
early-life cat allergen exposure and the FLG genotype on the
development of cat sensitization during childhood, and the effect
of early-life exposure was significantly greater among children
with FLG mutations (approximately 36% increase in risk per
log-unit increase in Fel d 1 in children with FLG mutations
compared with those without). FLG mutations significantly
increased the effect of early-life Der p 1 exposure on mite sensi-
tization at age 1 year, but this modifying effect was gradually
reduced over time. Markedly different patterns of the relationship
between FLG genotype and exposure to dog on sensitization were
observed in that the risk of dog sensitization appeared lower, and
the risk of sensitization to any allergen was significantly lower
among children with FLG mutations who were exposed to a
dog in infancy (on average, >5-fold reduction in the risk of sensi-
tization during childhood).
Limitations and strengths
The main limitation of our study is the lack of a replication
population. However, there are very few birth cohorts that have
objective measures of exposure to multiple allergens in early life
and that have assessed sensitization on multiple occasions from
early childhood to adolescence, both of which are key to
interpreting our findings. Also, we were unable to include all
cohort participants because of the availability of early-life dust
samples and FLG genotyping.
The 6 FLG mutations we assessed have been consistently asso-
ciated with eczema in white populations
36
; however, because
some of these mutations are not found in nonwhite subjects, all
nonwhite participants were excluded from our analyses, and the
results are not applicable to other ethnicities. Our definition of
loss-of-function mutations within the FLG gene included car-
rying 1 or more of the 6 genetic variations. As a result, among par-
ticipants with incomplete genotyping data in whom all alleles
successfully tested were wild-type, it was not possible to deter-
mine their FLG status, and these subjects were excluded from
further analysis, with a consequent reduction in sample size.
We repeated our analyses using FLG variants 2282del4 and
R501X only, and the results were entirely consistent with findings
when FLG status was defined by using all 6 mutations, with no
material change in any of the reported significant interactions
(data available on request).
Another limitation is the smaller sample size at age 1 year and
in some of the subgroups (eg, sensitized dog owners with FLG
TABLE I. Multivariable analysis indicating effect of the inter-
action between FLG loss-of-function mutations and Fel d 1,
Der p 1, and Can f 1 exposure on the risk of cat, mite, and dog
allergen-specific sensitization
Age Odds ratio 95% CI Pvalue
Interaction: FLG loss-off-function * Fel d 1 exposure, cat sensitization
1 y 12.57 0.00-489810.02 .64
3 y 1.48 0.81-2.73 .20
5 y 1.99 1.05-3.79 .035
8 y 1.59 1.07-2.37 .021
11 y 1.34 0.85-2.12 .20
16 y 1.07 0.72-1.61 .73
GEE: age 1-16 y 1.36 1.02-1.80 .035
GEE: complete age 3-16 y 1.76 1.09-2.83 .021
Interaction: FLG loss-off-function * Der p 1 exposure, dust mite
sensitization
1 y 0.00 0.00- . .99
3 y 1.00 0.60-1.65 .99
5 y 1.25 0.78-2.00 .36
8 y 0.97 0.64-1.46 .87
11 y 1.23 0.74-2.04 .42
16 y 0.93 0.54-1.60 .79
GEE: age 1-16 y 1.09 0.78-1.51 .62
GEE: complete age 3-16 y 0.91 0.53-1.56 .72
Interaction: FLG loss-off-function * Can f 1 exposure, dog sensitization
1 y 0.55 0.15-2.05 .37
3 y 0.79 0.42-1.49 .47
5 y 0.94 0.51-1.70 .83
8 y 0.75 0.24-2.41 .63
11 y 0.45 0.06-3.34 .43
16 y 0.77 0.32-1.88 .57
GEE: age 1-16 y 0.76 0.47-1.22 .26
GEE: complete age 3-16 y 1.08 0.50-2.36 .84
FLG genotype,allergen exposure, sex, breast-feeding, and socioeconomic status are
included as covariates.
J ALLERGY CLIN IMMUNOL
VOLUME 145, NUMBER 3
SIMPSON ET AL 997
mutations), and our findings need to be interpreted with caution.
We could not address the question about the relative importance
of exposure in infancy compared with that in later childhood.
The strengths of this study include comprehensive measure-
ments of early-life allergen exposure and objective evaluation
of sensitization from infancy to adolescence. Sensitization was
assessed at 6 time points, which allowed analysis of the effect
of allergen exposure and genotype over time. We used data on
both pet allergen exposure and pet ownership, and similar
findings in these 2 measures of exposure strengthen our
findings.
Interpretation
To our knowledge, this is the first study to investigate the
relationship between objectively measured exposure to inhalant
allergens and FLG mutations with longitudinal trajectories of
allergic sensitization. We have previously shown that FLG loss-
of-function mutations modify the effect of environmental peanut
exposure on the development of peanut sensitization and al-
lergy.
27
Our current study extends this to inhalant allergens and
suggests that the transcutaneous route through an impaired skin
barrier might be important for sensitization. However, although
there is currently no consensus about the presence of FLG in res-
piratory tissues,
37
we cannot exclude the possibility that the ef-
fects observed in this study are mediated through the inhaled
route and exposure in the nose because FLG might be expressed
in human nasal mucosa.
38
Two birth cohorts in the United Kingdom and Denmark have
shown a significant interaction between FLG loss-of-function
mutations and early-life cat ownership on the development of in-
fantile atopic dermatitis (AD).
26
In the birth cohort from The
Netherlands, early-life cat ownership enhanced the effect of
FLG mutations on AD at ages 4 and 8 years but, similar to our re-
sults, not on sensitization to any allergen.
39
A significant associ-
ation has been reported between the severity of AD and cat
sensitization in FLG-related AD but not in non–FLG-related
AD,
40
and one mechanism by which cat exposure could drive
the development of AD is by enhancing cat-specific sensitization
facilitated though an impaired skin barrier. In the current study in
children with wild-type FLG, Fel d 1 exposure increased the risk
of cat sensitization at age 1 year, but this association diminished
as children got older. In contrast, in children with FLG mutations,
the increased risk of cat sensitization related to high allergen
exposure in infancy persisted over time, with different trajectories
of sensitization during childhood in children with different
FIG 2. Effect of exposure to mite allergen Der p 1 on the predicted probability of mite-specific sensitization
among children with and without FLG loss-of-function mutations: ages 1, 3, 5, 8, 11, and 16 years. Ln, Nat-
ural log.
J ALLERGY CLIN IMMUNOL
MARCH 2020
998 SIMPSON ET AL
genotypes in relation to the same environmental exposure. By age
16 years, the point prevalence of cat-specific sensitization was the
same in children with and without a cat in both genotype groups.
This might be in part due to exposure to cat allergen outside the
home because previous studies have shown that cat allergen is
transported on clothing and can be measured at high levels in
homes without cats and public places.
33,41
FLG mutations significantly increased the effect of Der p 1
exposure on mite sensitization at age 1 year. After this, the inter-
action between FLG and exposure decreased considerably. It has
been shown that early sensitization (including that to mite and cat)
is crucially important for the development of asthma,
42-45
and our
finding that the interactions between FLG and cat and mite expo-
sure in relation to specific sensitizations are stronger in early life
might be one of the mechanisms by which FLG loss-of-function
mutations increase the risk of asthma.
24
Mite allergens have proteolytic activity,
46
which can disrupt the
skin barrier by cleaving tight junction proteins.
47
Thus mite aller-
gens might disrupt the skin barrier without the increased suscep-
tibility of FLG loss-of-function mutations. In support of this, in
BALB/c mice recombinant Der p 1 was able to induce eczema
without skin stripping or addition of an adjuvant.
48
However, it
is unclear whether the magnitude of exposure in the animal model
would resemble skin exposure in infants in real life, and there are
no definitive studies to confirm this.
The effect of exposure to dogs differed from that observed for
cats and mites in that dog ownership (and high exposure to dog
allergen) was protective among children with FLG loss-of-
function mutations and that this protective effect extended to
sensitization to any allergen. Dog ownership can offer protection
through the increase in microbial exposure,
1,49
and our finding of
a significantly stronger protective effect among subjects with
FLG mutations can be explained by comparatively greater per-
sonal exposure to microbial products consequent to the impaired
skin barrier. The fact that significant effects of Fel d 1 exposure
and cat ownership were confined to cat-specific sensitization
(but not sensitization to other allergens) suggests that the
observed effects are related to allergen exposure. Taken together,
our data support the notion that differences in the effects of cat
and dog ownership might be a consequence of a cat being a
marker of high allergen exposure, whereas the protective effect
of dog exposure can be mediated through changes in the skin
microbiome.
50
The findings of the current study confirm our previous
observation of the changing nature of the association between
early-life exposures and sensitization with time and the crucial
FIG 3. Effect of exposure to dog allergen Can f 1 on the predicted probability of dog-specific sensitization
among children with and without FLG loss-of-function mutations: age 1, 3, 5, 8, 11, and 16 years. Ln, Natural
log.
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VOLUME 145, NUMBER 3
SIMPSON ET AL 999
importance of longitudinal analyses.
15
Furthermore, they raise
fundamental questions about the current approach to replication
in genetic and gene-environment studies because the timing of
the assessment of outcomes can critically affect the results of
different studies investigating genes, the environment, and their
interactions. Where there are inconsistencies, we should move
from direct replication toward triangulation (ie, integration of ev-
idence from several approaches with differing and unrelated sour-
ces of bias) to improve causal inference.
51
In conclusion, FLG loss-of-function mutations modify the rela-
tionship between early-life allergen exposure and sensitization,
but effects differ at different ages and between different allergens.
Children with FLG mutations might benefit from mite and cat
avoidance but can gain from having a dog in early life, but this
will have to be confirmed in prospective studies.
We thank the study participants and their parents for their continued support
and enthusiasm. We greatly appreciate the commitment they have given to the
project. We would also like to acknowledge the hard work and dedication of
the study teams (postdoctoral scientists, physiologists, research fellows,
nurses, technicians, and clerical staff). Dr Jenny Hankinson completed the
FLG genotyping.
Clinical implications: Children with FLG mutations might
benefit from mite and cat avoidance but might gain from having
a dog in early life.
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