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\begin{document}

\title{Minimal changes in the B- and T-cell compartments of school-aged
children with haploinsufficiency of filaggrin: The Generation R Study}



\author[1]{Kirsten Looman}%
\author[2]{Minke van Mierlo}%
\author[3]{Menno van Zelm}%
\author[4]{Chen Hu}%
\author[5]{Liesbeth Duijts}%
\author[2]{Johan de Jongste}%
\author[2]{Tamar Nijsten}%
\author[6]{Luba Pardo}%
\author[7]{Jessica Kiefte}%
\author[5]{Henriëtte Moll}%
\author[2]{Suzanne Pasmans}%
\affil[1]{Erasmus University Medical Center}%
\affil[2]{Erasmus MC}%
\affil[3]{Central Clinical School, Monash University and Alfred Hospital}%
\affil[4]{Erasmus Universiteit Rotterdam}%
\affil[5]{Erasmus Medical Center}%
\affil[6]{Erasmus MC University Medical Center Rotterdam}%
\affil[7]{LUMC}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Mutations in the filaggrin gene (FLG) affect epidermal
barrier function and increase the risk of atopic dermatitis (AD). We
hypothesized that FLG mutations affect immune cell composition in a
general pediatric population. Therefore, we investigated if school-aged
children with and without FLG mutations have differences in immune cell
numbers. Methods: This study was embedded in a population-based
prospective cohort study, the Generation R Study, and included 523
children of European genetic ancestry aged 10 years. The most common FLG
mutations in the European population (R501X, S1085CfsX36, R2447X and
S3247X) were genotyped. Additionally, 11-color flow cytometry was
performed on peripheral blood samples to determine helper T (Th),
regulatory T (Treg) and CD27+ and CD27- memory B cells. Sensitivity
analysis was performed in 102 AD cases, assessed by parental
questionnaires. Results: FLG mutations were observed in 8.4\% of the
total population and in 15.7\% of the AD cases. Children with any FLG
mutation had higher Th22 cell numbers compared to FLG wild-type
children. Children with and without FLG mutations had no difference in
Th1, Th2, Th17, Treg or memory B cell numbers. Furthermore, in children
with AD, FLG mutation carriership was not associated with differences in
T- and B-cells or their subsets. Conclusions: School-aged children with
FLG mutations have higher Th22 cell, which might suggest an
immunological defense mechanism to an altered skin barrier function. No
associations between Th or Treg cells and FLG mutations suggests that
FLG mutations do not otherwise affect immune composition in a general
pediatric population.%
\end{abstract}%



\sloppy


\textbf{TITLE PAGE}

\textbf{Title}

Minimal changes in the B- and T-cell compartments of school-aged
children with haploinsufficiency of filaggrin: The Generation R Study

\textbf{Short Title}

Filaggrin haploinsufficiency and B and T cell subsets

\textbf{Word count:} 2488

\textbf{Number of figures:} 3

\textbf{Number of tables:} 1

\textbf{Electronic repository:} 1 table, 2 figures

\textbf{Author names and affiliations}

Kirsten I.M. Looman\textsuperscript{1,2}*, Minke M.F. van
Mierlo\textsuperscript{3}*, Menno C. van Zelm\textsuperscript{4}, Chen
Hu\textsuperscript{1,3}, Liesbeth Duijts\textsuperscript{5,6}, Johan C.
de Jongste\textsuperscript{1,5}, Tamar Nijsten\textsuperscript{3}, Luba
M. Pardo\textsuperscript{3}, Jessica C. Kiefte-de
Jong\textsuperscript{7}, Henri\selectlanguage{ngerman}ëtte A. Moll\textsuperscript{2}, Suzanne
G.M.A. Pasmans\textsuperscript{3}

\emph{* Authors denote equal contribution.}

\textsuperscript{1} The Generation R Study Group, Erasmus MC, University
Medical Center Rotterdam, Rotterdam, the Netherlands.\textsuperscript{2}
Department of General Pediatrics, Erasmus MC, University Medical Center
Rotterdam- Sophia Children's Hospital, Rotterdam, the
Netherlands.\textsuperscript{3} Department of Dermatology, Erasmus MC,
University Medical Center Rotterdam-Sophia Children's Hospital-Center of
Pediatric Dermatology, Rotterdam, the Netherlands.\textsuperscript{4}
Department Immunology and Pathology, Central Clinical School, Monash
University and The Alfred Hospital, Melbourne, VIC,
Australia.\textsuperscript{5} Department of Pediatrics, Division of
Respiratory Medicine and Allergology, Erasmus MC, University Medical
Center Rotterdam, Rotterdam, the Netherlands.\textsuperscript{6}
Department of Pediatrics, Division of Neonatology, Erasmus MC,
University Medical Center Rotterdam, Rotterdam, the
Netherlands.\textsuperscript{7} Department of Public Health and Primary
Care, Leiden University Medical Center/LUMC Campus, The Hague, The
Netherlands.

\textbf{Corresponding author:}

Suzanne G.M.A. Pasmans, Department of Dermatology\selectlanguage{english}-Center of Pediatric
Dermatology, Erasmus MC University Medical Center Rotterdam- Sophia
Children's Hospital, Sp\selectlanguage{english}-1540, Wytemaweg 80, 3015 CN Rotterdam, The
Netherlands.

\textbf{Conflict of Interest Statement:}

The Generation R Study is conducted by Erasmus MC University Medical
Center Rotterdam in close collaboration with the School of Law and
Faculty of Social Sciences of Erasmus University Rotterdam, the
Municipal Health Service Rotterdam Metropolitan Area, the Rotterdam
Homecare Foundation, and the Stichting Trombosedienst \&
Artsenlaboratorium Rijnmond. The Dept Immunology of the Erasmus MC
funded the immunological measurements. LD received funding from the
European Union's Horizon 2020 research and innovation programme
(LIFECYCLE, grant agreement No 733206, 2016; EUCAN-Connect grant
agreement No 824989; ATHLETE, grant agreement No 874583). MCvZ is
supported by the Australian National Health and Medical Research Council
(NHMRC, Senior Research Fellowship 1117687). All authors declare that no
competing interests exist. The Department of Dermatology of the Erasmus
MC University Medical Center Rotterdam received an unrestricted grant
from Micreos Human Health, the Netherlands.

\textbf{Funding statement}

LIFECYCLE, grant agreement No 733206, 2016; EUCAN-Connect grant
agreement No 824989; ATHLETE, grant agreement No 874583; Australian
National Health and Medical Research Council, Grant/Award Number:
1117687; European Union's Horizon 2020 research and innovation
programme, Grant/Award Number: 733206; European Union's Horizon 2020
co\selectlanguage{english}-funded programme ERA\selectlanguage{english}-Net on Biomarkers for Nutrition and Health (ERA
HDHL), Grant/Award Number: 696295. The Department of Dermatology of the
Erasmus MC University Medical Center Rotterdam received an unrestricted
grant from Micreos Human Health, The Netherlands.

\textbf{ABSTRACT}

\textbf{Background:} Mutations in the filaggrin gene (\emph{FLG} )
affect epidermal barrier function and increase the risk of atopic
dermatitis (AD). We hypothesized that \emph{FLG} mutations affect immune
cell composition in a general pediatric population. Therefore, we
investigated if school-aged children with and without
\emph{FLG}mutations have differences in immune cell numbers.

\textbf{Methods:} This study was embedded in a population-based
prospective cohort study, the Generation R Study, and included 523
children of European genetic ancestry aged 10 years. The most
common\emph{FLG} mutations in the European population (R501X,
S1085CfsX36, R2447X and S3247X) were genotyped. Additionally, 11-color
flow cytometry was performed on peripheral blood samples to determine
helper T (Th), regulatory T (Treg) and CD27\textsuperscript{+} and
CD27\textsuperscript{-} memory B cells. Sensitivity analysis was
performed in 102 AD cases, assessed by parental questionnaires.

\textbf{Results:} \emph{FLG} mutations were observed in 8.4\% of the
total population and in 15.7\% of the AD cases. Children with
any\emph{FLG} mutation had higher Th22 cell numbers compared to
\emph{FLG}wild-type children. Children with and without \emph{FLG}
mutations had no difference in Th1, Th2, Th17, Treg or memory B cell
numbers. Furthermore, in children with AD, \emph{FLG} mutation
carriership was not associated with differences in T- and B-cells or
their subsets.

\textbf{Conclusions:} School-aged children with \emph{FLG} mutations
have higher Th22 cell, which might suggest an immunological defense
mechanism to an altered skin barrier function. No associations between
Th or Treg cells and \emph{FLG} mutations suggests that
\emph{FLG}mutations do not otherwise affect immune composition in a
general pediatric population.

\textbf{KEYS MESSAGE}

This study in a general pediatric population showed that filaggrin
haploinsufficiency is associated with higher Th22, but not with other B-
and T-cell subsets. \emph{FLG} mutations did not affect immune cell
composition in a subset of children with AD. Therefore, FLG mutations
pose a risk to allergy development, but do not directly affect or
confound immune measurements in children with atopic dermatitis.

\textbf{INTRODUCTION}

Filaggrin is a filament-associated protein that is encoded by the
Filaggrin gene (\emph{FLG} ),\textsuperscript{1} and is an important
contributor to the preservation of the skin
barrier.\textsuperscript{1,2} Approximately 10\% of the European
population is heterozygote carrier of a disrupting mutation in\emph{FLG}
.\textsuperscript{3} Both complete loss-of-function and reduced
functional activity of filaggrin lead to destruction of the stratum
corneum (SC) and to skin barrier dysfunction.\textsuperscript{2,4} This
barrier dysfunction due to\emph{FLG} mutations is presumed to be caused
by lower numbers of tight junctions, reduced density of the protein
corneodesmosin and impaired maturation and excretion of lamellar bodies
in the epidermis which are important in maintaining cell-to-cell
integrity.\textsuperscript{1}

Failure in barrier function through mutations in \emph{FLG} results in
increased skin permeability for percutaneous transfer of exogenous
particles including allergens and pollutants including an increased
permeability of the skin for percutaneous protein
transfer.\textsuperscript{1,2,4} Accordingly, \emph{FLG} mutations are
the strongest genetic risk factor for atopic dermatitis (AD),
predisposing to a form of AD that starts in early infancy and persists
into adulthood.\textsuperscript{2,3,5} Previous meta-analysis showed
that \emph{FLG} haploinsuffiency results in an odds ratio (OR) of 3.12
for AD.\textsuperscript{6} In addition, \emph{FLG} mutations in AD are
associated with a higher incidence of skin infections or superinfections
such as eczema herpeticum and a higher likelihood of having asthma,
inhalant or food allergies.\textsuperscript{1,7-9}

The increased permeability of the skin as a result of
\emph{FLG}mutations is thought to affect immune responses and maturation
of adaptive immune cells. Filaggrin is also expressed in the thymus, the
primary lymphoid organ in which T cells are
formed.\textsuperscript{10}Hence, \emph{FLG} mutations potentially
affect the peripheral immune cell compartment through effects in skin
and thymus, and previous studies observed higher \selectlanguage{greek}γδ\selectlanguage{english}T17, T helper (Th) 17
in mice bi-allelic for\emph{FLG} disruption.\textsuperscript{10} A case
study reported higher numbers of circulating thymus-emigrated regulatory
T (Treg) cells, Th2 in with 6 AD patients with a heterozygote
\emph{FLG}mutation.\textsuperscript{11} Another study with 2
heterozygous, 2 homozygous and 1 compound heterozygous AD patient showed
increased Th17 cells in \emph{FLG} -mutation group.\textsuperscript{12}
On the other hand, literature on the role of B cell dysregulation in AD
is scarce and conflicting.\textsuperscript{13-16}. It can be
hypothesized that mutations in \emph{FLG} can affect B-cell numbers due
to skewing of the Th-cell population.

Because of the function of filaggrin in skin barrier tissue and in
thymus, the presence of \emph{FLG} mutations might be a contributor to
the shaping of T -and B cell maturation in children. However, no studies
on this association have been performed in the general pediatric
population and only case studies have been performed in AD
patients.\textsuperscript{11,12} Therefore, we here studied the
associations between common \emph{FLG} mutations in the European
population and immune cell numbers, as determined using with 11-color
flow cytometry, within a population-based birth cohort study including a
subgroup of AD patients.

\textbf{METHODS}

\textbf{Study design}

This study was embedded within the Generation R Study, a prospective
birth cohort study located in Rotterdam, the Netherlands. The Medical
Ethical Committee of the Erasmus MC, University Medical Center Rotterdam
approved the study (MEC-2012-165).\textsuperscript{17} Written informed
consent was obtained from parents or legal representatives of all
children. We included all children with European genetic
ancestry\textsuperscript{18} with information on filaggrin mutation
(homozygous, compound heterozygous, heterozygous or wild type) and
information on at least one of the immune cell outcomes. This resulted
in a total number of 523 children (Figure 1).

Our subgroup of children with AD, defined as physician-diagnosed eczema
from parental questionnaires obtained at the child's age of 10 years
(`Was your child ever diagnosed by a physician with atopic dermatitis',
`yes; no') consisted of 102 subjects.\textsuperscript{16}

\emph{FLG} \textbf{genotype}

DNA samples obtained from umbilical cord blood were genotyped by
modified Taqman allelic discrimination assays with the use of primers as
described previously.\textsuperscript{19} The following 4
\emph{FLG}mutations were identified: R501X (rs61816761), S1085CfsX36
(rs41370446), R2447X (rs138726443), and S3247X
(rs150597413).\textsuperscript{19,20}Children were classified as having
a \emph{FLG} mutation if they were homozygous, compound heterozygous or
heterozygous for any of the four mutations. Children without any of the
four mutations were classified as wild type.

\textbf{Immune cell numbers}

Peripheral blood samples from children were obtained at the age of 10
years.\textsuperscript{16} Absolute counts of CD3\textsuperscript{+} T
cells and CD19\textsuperscript{+} B cells per \selectlanguage{greek}μ\selectlanguage{english}Lblood were determined
with diagnostic lyse-no-wash protocol and detailed immunophenotyping was
performed with 11-color flow cytometry (LSRII Fortessa, BD Biosciences).
We determined naive (CD45RO\textsuperscript{-}CCR7\textsuperscript{+}),
effector memory RO-positive T cells (TemRO;
CD45RO\textsuperscript{+}CCR7\textsuperscript{-}) and effector memory
RA-positive T cells (TemRA;
CD45RO\textsuperscript{-}CCR7\textsuperscript{-}) within
CD4\textsuperscript{+} and
CD8\textsuperscript{+}lineages.\textsuperscript{16,21,22} Within Treg
cells, the differentiation in naive (CD45RA\textsuperscript{+}) and
memory (CD45RA\textsuperscript{-}) was determined.\textsuperscript{16}
Finally, the following T helper (Th) cell subsets
(CD4\textsuperscript{+}CD45RA\textsuperscript{-}) were determined after
exclusion of Treg cells: Th1
(CCR6\textsuperscript{-}CXCR3\textsuperscript{+}CCR4\textsuperscript{-}),
Th2
(CCR6\textsuperscript{-}CXCR3\textsuperscript{-}CCR4\textsuperscript{+}),
Th17
(CCR6\textsuperscript{+}CXCR3\textsuperscript{-}CCR4\textsuperscript{+}CCR10\textsuperscript{-}),
Th17.1(CCR6\textsuperscript{+}CXCR3\textsuperscript{+}CCR4\textsuperscript{--})
and Th22
(CCR6\textsuperscript{+}CXCR3\textsuperscript{--}CCR4\textsuperscript{+}CCR10\textsuperscript{+}).s
In addition CD27\textsuperscript{+} and
CD27\textsuperscript{-}IgG\textsuperscript{+}, IgA\textsuperscript{+},
IgE\textsuperscript{+}CD19\textsuperscript{+}CD38\textsuperscript{dim}IgD\textsuperscript{-}memory
B cell subsets were defined.\textsuperscript{16} Gating strategies for
immune cell determination were published previously.\textsuperscript{16}

\textbf{Statistical Analyses}

First, characteristics of the study population were determined,
stratified for \emph{FLG} mutations . P-values for determining
differences between the categorical variables between the two groups
were calculated with chi-squared tests. Next, median cell numbers with
interquartile range (IQR) were determined. Differences in cell numbers
between children with and without \emph{FLG} mutations were determined
with the non-parametric Mann-Whitney U tests. Sensitivity analyses on
the associations of \emph{FLG} genotype with immune cell numbers were
performed within children who were diagnosed with AD. No adjustment for
multiple testing was performed because of strong correlation between the
immune cells studied. Statistical analyses were performed with SPSS
version 21.0 (IBM Corp.) and R version 3.3.3 (R Foundation for
Statistical Computing).

\textbf{RESULTS}

\textbf{Study population}

Characteristics of the study population are presented in Table 1. Within
the total group of 523 children with European ancestry, filaggrin
mutations were detected in 44 (8.4\%) children, including 3 biallelic
mutations (2 compound heterozygous and 1 homozygous). The proportion of
patients with AD was lower in the wild-type group compared to the group
with \emph{FLG} mutations (20\% versus 42\%; p\textless{}0.01). Within
the atopic dermatitis population, 15.7\% children had a
\emph{FLG}mutation, including one biallelic mutation (homozygous). No
differences were observed between other atopic diseases, including
asthma, inhalant and food allergy and \emph{FLG} mutation status (data
not shown).

\textbf{Higher Th22 cell counts in children with \emph{FLG} mutations}

Children with \emph{FLG} mutation had higher Th22 cell numbers compared
to children of the wild-type population (Figure 2A, eTable1). The median
cell number within the \emph{FLG} mutation group was 5.60/\selectlanguage{greek}μ\selectlanguage{english}L (IQR
4.04;8.94) and within the wild-type group 4.54/\selectlanguage{greek}μ\selectlanguage{english}L (IQR 2.50;7.38, p
=0.03). In contrast, when studying this association in the atopic
dermatitis population, no significant associations were observed
(eFigure 1A).

\textbf{No associations between Th1, Th2, Th17 and Treg and
\emph{FLG}mutations}

No differences in median cell numbers between the \emph{FLG} mutation
and wild type group were observed for Th1, Th2, Th17 and Treg (Figure
2A, eTable 1). This was similar in the sensitivity analyses that only
included children with atopic dermatitis (eFigure 1A). No differences in
median cell numbers between the \emph{FLG} mutation and the wild type
group were observed for the effector memory CD4\textsuperscript{+} and
CD8\textsuperscript{+} T cell subsets: naive, Tcm, TemRA, TemRO (Figure
2B, eFigure 1B).

\textbf{No associations between memory B cells and \emph{FLG} mutations}

No differences in median cell numbers between the \emph{FLG} mutation
and the wild type group were observed for total B cells and naive mature
B cells (eTable 1). In addition, no associations between
\emph{FLG}mutations and the following CD27\textsuperscript{+} and
CD27\textsuperscript{-} memory B cell subsets were observed:
IgA\textsuperscript{+}, IgE\textsuperscript{+}, IgG\textsuperscript{+},
IgM\textsuperscript{+} (Figure 3). Similarly, no changes were observed
in the sensitivity analyses, which only included children with atopic
dermatitis (eFigure 2, eTable 1).

\textbf{DISCUSSION}

In this population-based study among children of European genetic
ancestry, we observed a prevalence of 8.4\% for \emph{FLG} mutations. In
addition, we demonstrated that children with \emph{FLG} mutations had
higher Th22 cell numbers than children without \emph{FLG} mutations. In
contrast, the Th1, Th2, Th17, Treg and memory B cell numbers were
comparable between children with and without \emph{FLG} mutations. In
addition, among children with AD, those with or without
\emph{FLG}mutations had no differences in B or T cell subsets.

\textbf{Comparison with literature and interpretation}

All previous studies on \emph{FLG} mutations and immune cell numbers
have been performed within mice models or smaller numbers of AD
patients.\textsuperscript{10-12,23} This is the first study that
provides insight in the role of \emph{FLG} mutations on immune cell
numbers in school-aged children of a general population. The setting of
this study within a population-based pediatric cohort study is unique to
study the association of \emph{FLG} on immune cell numbers in a general
population.

In line with a previous study in mice, we observed higher Th22 cell
numbers in children with \emph{FLG} mutations than in to children with
wild type \emph{FLG} alleles. \textsuperscript{23} Th22 cells are
characterized by the production of IL-22 and contributes to skin
integrity.\textsuperscript{24,25} Moreover, IL-22 is known for its role
in the defense of different pathogens in the skin by the production of
antimicrobial proteins.\textsuperscript{24,25} In AD, acute skin lesions
are associated with an upregulated Th22 immune
response.\textsuperscript{26,27} We speculate that the observed higher
number of Th22 cells is an immunological defense mechanism to the
damaged skin barrier. Nevertheless, future studies should confirm this
finding. When studying Th22 numbers only in AD children, no differences
were observed between both groups. This is could be explained by the
fact that children with AD have higher Th22 independent of
their\emph{FLG} genotype. \textsuperscript{26} In addition, the
expression of the protein filaggrin is decreased in AD which can cause
Th22 alterations through skin barrier dysfunction independent of
\emph{FLG}mutation status.\textsuperscript{1,5} Unfortunately,
literature on the effect of \emph{FLG} mutations on the numbers of Th22
in AD is lacking.

In contrast with small previous case studies in AD, we did not observe
differences in Th2, Th17 and Treg cell numbers between children with and
without \emph{FLG} mutations both in the total study population and in
the subgroup of patients with AD. \textsuperscript{10-12,23} The
associations of Th2, Th17 and Treg cell numbers with filaggrin in
previous studies were explained by increased TSLP activity due to
impaired skin barrier and impaired thymus
functioning.\textsuperscript{11,28} In the thymus, an altered filaggrin
expression affects maturation of T cells and induces dendritic cells to
stimulate the differentiation of naive Treg cells into functionally
different Treg cells. \textsuperscript{10,29} The discrepancies between
previous studies and our current study could be explained by differences
in investigated populations and species. The mice studies
studied\emph{FLG} null mutations, in which no expression of filaggrin is
present.\textsuperscript{10} This is in contrast with our population,
encompassing mainly haploinsufficient children which leads to a 50\%
reduction in filaggrin expression. \textsuperscript{1} In
addition,\emph{FLG} knockdown skin equivalents in previous studies could
represent a different immunological setting than is present in human
skin. \textsuperscript{23} Furthermore, it is likely that previous
results on immune cell numbers in AD populations are affected by disease
severity. Namely, immune cells in active AD skin can induce
downregulation of filaggrin protein expression in the skin independent
of \emph{FLG} mutations, subsequently affecting immune cell composition.
Although we do not have information on disease severity in our AD
population, this study included a relatively healthy cohort in which we
expect relatively mild AD. A more severe AD phenotype has been linked to
homozygous and compound heterozygous \emph{FLG} mutation
phenotype.\textsuperscript{1} In addition, activated immune cells in
more active AD skin can in itself downregulate filaggrin protein
expression in the skin independent of \emph{FLG} mutations and thereby
affect skin barrier function and subsequently immune cell
alteration.\textsuperscript{1,5}In accordance, a previous study in AD
patients observed that circulating Treg cell numbers were associated
with AD activity and not with\emph{FLG} genotype.\textsuperscript{11} In
addition, we previously showed that children with AD had higher Th17 and
Treg memory cell numbers.\textsuperscript{16} Therefore, alteration in
immune cell numbers is probably not only dependent on \emph{FLG}
mutation genotype, but also on AD severity and epigenetic and
environmental factors that affect these two, as our data suggest.

In addition, we did not observe differences in memory B cell numbers
between children with and without \emph{FLG} mutations, nor any
associations between memory B cell numbers and AD in our previous
study.\textsuperscript{16} No previous studies have investigated B cell
subsets in relation to \emph{FLG} mutations. Possibly, T cells are more
affected by \emph{FLG} mutations than B cells because of the effects of
filaggrin in the thymus.\textsuperscript{10}

\textbf{Methodological considerations}

A major strength is that this study investigated the association
between\emph{FLG} genotype and a large panel of B and T cells in the
general population for the first time. We had detailed and extensive
information on immune cell numbers from 11-color flow cytometry and
obtained objective information on genetic ancestry. However, the
following limitations need to be addressed. First, the AD population for
the sensitivity analyses was relatively small which could have limited
the power in the statistical analyses. Nevertheless, in comparison to
previous studies, only including a maximum of 6 AD patients
with\emph{FLG} mutations, this is the largest study on \emph{FLG}
mutations in both the general population and AD patients. Second, our AD
population was defined by ever having physician diagnosed AD before or
at the age of 10 years. Therefore, it is likely that a subset of the
children has outgrown AD at the age of 10 and this might affect their
immunophenotype. In addition, the use of questionnaires for AD diagnosis
could have introduced recall bias. Third, as mentioned previously, our
study included the four most common \emph{FLG} mutations in the European
population. To prevent misclassification, we selected children with
genetic European ancestry for the current study. Although the choice for
including the most common \emph{FLG} mutations in European populations
is in line with previous studies \textsuperscript{11,12}, other less
frequent \emph{FLG} mutations could exist in low numbers since up to
113\emph{FLG} mutations resulting in premature protein termination have
been described. A recent study including patients with AD and Ichthyosis
Vulgaris (IV), showed that screening the entire encoding region
of\emph{FLG} for mutations led to an improvement of the diagnostic
yield.\textsuperscript{30} Furthermore, biallelic genotypes were only
present in 3 children of the study population and therefore not studied
separately. Since homozygous or compound heterozygous mutations lead to
complete absence of filaggrin expression, these mutations might have
different effects on immune cell numbers compared to
heterozygous\emph{FLG} mutations which lead to 50\% reduction in
filaggrin expression.\textsuperscript{1} Therefore, studies
investigating biallelic mutations compared to heterozygous \emph{FLG}
mutations and wild type \emph{FLG} are needed. Furthermore, as this is
the first study in a general cohort addressing the association between
\emph{FLG}mutation and immune cell numbers, future studies are needed
for validation of our results.

In conclusion, within the general population, school-aged children
with\emph{FLG} mutations have higher Th22 cell numbers. Furthermore,
children with and without \emph{FLG} haploinsufficiency did not differ
in other B and T cell subsets. This suggests that with age, other
factors such as environmental exposure contribute to risk that\emph{FLG}
mutations poses to the development of allergy.

\textbf{ACKNOWLEDGMENTS}

We gratefully acknowledge the contribution of children and parents,
general practitioners, hospitals and midwives, and pharmacies in
pharmacies in Rotterdam. We thank the following parties for financially
supporting our work: IFECYCLE, grant agreement No 733206, 2016;
EUCAN-Connect grant agreement No 824989; ATHLETE, grant agreement No
874583; Australian National Health and Medical Research Council,
Grant/Award Number: 1117687; European Union's Horizon 2020 research and
innovation programme, Grant/Award Number: 733206; European Union's
Horizon 2020 co-funded programme ERA-Net on Biomarkers for Nutrition and
Health (ERA HDHL), Grant/Award Number: 696295. The Department of
Dermatology of the Erasmus MC University Medical Center Rotterdam
received an unrestricted grant from Micreos Human Health, The
Netherlands. The funders had no role in the design or conduct of the
study; the collection of the data and analyses; the interpretation of
data; the preparation and review or approval of the manuscript; or the
decision to submit the manuscript.

\textbf{ETHICAL APPROVAL STATEMENT}

The study was conducted according to the principles of the Declaration
of Helsinki. The Medical Ethical Committee of the Erasmus MC, University
Medical Center Rotterdam approved the study (MEC-2012-165). Written
informed consent was obtained from parents or legal representatives of
all children.

\textbf{REFERENCES}

1. Irvine AD, McLean WH, Leung DY. Filaggrin mutations associated with
skin and allergic diseases. \emph{N Engl J Med.} 2011;365(14):1315-1327.

2. Sandilands A, Sutherland C, Irvine AD, McLean WH. Filaggrin in the
frontline: role in skin barrier function and disease. \emph{J Cell
Sci.}2009;122(Pt 9):1285-1294.

3. Thyssen JP, Elias PM. It Remains Unknown Whether Filaggrin Gene
Mutations Evolved to Increase Cutaneous Synthesis of Vitamin
D.\emph{Genome Biol Evol.} 2017;9(4):900-901.

4. Goleva E, Berdyshev E, Leung DY. Epithelial barrier repair and
prevention of allergy. \emph{J Clin Invest.} 2019;129(4):1463-1474.

5. McAleer MA, Irvine AD. The multifunctional role of filaggrin in
allergic skin disease. \emph{J Allergy Clin
Immunol.}2013;131(2):280-291.

6. Rodriguez E, Baurecht H, Herberich E, et al. Meta-analysis of
filaggrin polymorphisms in eczema and asthma: robust risk factors in
atopic disease. \emph{J Allergy Clin Immunol.} 2009;123(6):1361-1370
e1367.

7. Brown SJ, Asai Y, Cordell HJ, et al. Loss-of-function variants in the
filaggrin gene are a significant risk factor for peanut allergy. \emph{J
Allergy Clin Immunol.} 2011;127(3):661-667.

8. Rice NE, Patel BD, Lang IA, et al. Filaggrin gene mutations are
associated with asthma and eczema in later life. \emph{J Allergy Clin
Immunol.} 2008;122(4):834-836.

9. Chan A, Terry W, Zhang H, et al. Filaggrin mutations increase
allergic airway disease in childhood and adolescence through
interactions with eczema and aeroallergen sensitization. \emph{Clin Exp
Allergy.} 2018;48(2):147-155.

10. Jee MH, Johansen JD, Buus TB, et al. Increased Production of
IL-17A-Producing gammadelta T Cells in the Thymus of Filaggrin-Deficient
Mice. \emph{Front Immunol.} 2018;9:988.

11. Moosbrugger-Martinz V, Gruber R, Ladstatter K, et al. Filaggrin null
mutations are associated with altered circulating Tregs in atopic
dermatitis. \emph{J Cell Mol Med.} 2019;23(2):1288-1299.

12. Bonefeld CM, Petersen TH, Bandier J, et al. Epidermal filaggrin
deficiency mediates increased systemic T-helper 17 immune
response.\emph{Br J Dermatol.} 2016;175(4):706-712.

13. Czarnowicki T, Esaki H, Gonzalez J, et al. Alterations in B-cell
subsets in pediatric patients with early atopic dermatitis. \emph{J
Allergy Clin Immunol.} 2017;140(1):134-144 e139.

14. Czarnowicki T, Gonzalez J, Bonifacio KM, et al. Diverse activation
and differentiation of multiple B-cell subsets in patients with atopic
dermatitis but not in patients with psoriasis. \emph{J Allergy Clin
Immunol.} 2016;137(1):118-129 e115.

15. Heeringa JJ, Hajdarbegovic E, Thio HB, van Zelm MC. Systemic B-cell
abnormalities in patients with atopic dermatitis? \emph{J Allergy Clin
Immunol.} 2016;138(1):317-318.

16. Looman KIM, van Meel ER, Grosserichter-Wagener C, et al.
Associations of Th2, Th17, Treg cells, and IgA(+) memory B cells with
atopic disease in children: The Generation R Study.
\emph{Allergy.}2020;75(1):178-187.

17. Kooijman MN, Kruithof CJ, van Duijn CM, et al. The Generation R
Study: design and cohort update 2017. \emph{Eur J
Epidemiol.}2016;31(12):1243-1264.

18. Medina-Gomez C, Felix JF, Estrada K, et al. Challenges in conducting
genome-wide association studies in highly admixed multi-ethnic
populations: the Generation R Study. \emph{Eur J
Epidemiol.}2015;30(4):317-330.

19. Elbert NJ, Duijts L, den Dekker HT, et al. Role of environmental
exposures and filaggrin mutations on associations of ethnic origin with
risk of childhood eczema. The Generation R Study. \emph{Pediatr Allergy
Immunol.} 2016;27(6):627-635.

20. Kezic S, O'Regan GM, Yau N, et al. Levels of filaggrin degradation
products are influenced by both filaggrin genotype and atopic dermatitis
severity. \emph{Allergy.} 2011;66(7):934-940.

21. Appay V, van Lier RA, Sallusto F, Roederer M. Phenotype and function
of human T lymphocyte subsets: consensus and issues. \emph{Cytometry
A.}2008;73(11):975-983.

22. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets
of memory T lymphocytes with distinct homing potentials and effector
functions. \emph{Nature.} 1999;401(6754):708-712.

23. Wallmeyer L, Dietert K, Sochorova M, et al. TSLP is a direct trigger
for T cell migration in filaggrin-deficient skin equivalents. \emph{Sci
Rep.} 2017;7(1):774.

24. Eyerich K, Eyerich S. Th22 cells in allergic disease. \emph{Allergo
J Int.} 2015;24(1):1-7.

25. Fujita H. The role of IL-22 and Th22 cells in human skin
diseases.\emph{J Dermatol Sci.} 2013;72(1):3-8.

26. Czarnowicki T, He H, Krueger JG, Guttman-Yassky E. Atopic dermatitis
endotypes and implications for targeted therapeutics. \emph{J Allergy
Clin Immunol.} 2019;143(1):1-11.

27. Jiao Q, Qian Q, Liu C, et al. T helper 22 cells from Han Chinese
patients with atopic dermatitis exhibit high expression of inducible
T-cell costimulator. \emph{Br J Dermatol.} 2020;182(3):648-657.

28. Chang J, Mitra N, Hoffstad O, Margolis DJ. Association of Filaggrin
Loss of Function and Thymic Stromal Lymphopoietin Variation With
Treatment Use in Pediatric Atopic Dermatitis. \emph{JAMA
Dermatol.}2017;153(3):275-281.

29. Tsilingiri K, Fornasa G, Rescigno M. Thymic Stromal Lymphopoietin:
To Cut a Long Story Short. \emph{Cell Mol Gastroenterol
Hepatol.}2017;3(2):174-182.

30. van Leersum FS, Nagtzaam IF, van Oosterhoud CN, et al. Improving the
diagnostic yield for filaggrin; concealed mutations in the Dutch
population. \emph{J Allergy Clin Immunol.} 2020;145(6):1704-1706 e1702.



\textbf{TABLES}

\textbf{Table 1. Descriptives of the study population}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Total population}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Total population}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Total population}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Total population}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Sensitivity analyses}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Child characteristics}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Total (n=523)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Wildtype population} \textbf{(n=479)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{FLG} \textbf{mutation population (n=44)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{P-value}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Missing in total study population (N, \%)}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Atopic dermatitis (n=102)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Sex (N, \%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
1.0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Female\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
280 (53.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
256 (53.4)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
24 (54.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
48 (47.1)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Male\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
243 (46.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
223 (46.6)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
20 (45.5)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
54 (52.9)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{FLG} mutations (N, \%)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Wildtype\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
479 (91.6)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
479 (100.0)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
86 (84.3)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1 or more mutations\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
44 (8.4) +\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
44 (100.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
16 (15.7)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Type \emph{FLG} mutations (N, \%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
S1085CfsX36 (rs41370446)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
22 (4.2)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
22 (4.2)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7 (6.9)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
R2447X (rs138726443)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6 (1.1)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6 (1.1)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1 (1.0)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
R501X (rs61816761)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
18 (3.5)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
18 (3.5)\textsuperscript{2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9 (9.0) ++\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
S3247X (rs150597413)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0 (0.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0 (0.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0 (0.0)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Ever physician diagnosed atopic dermatitis (N, \%)\selectlanguage{ngerman}§\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
102 (22.2)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
86 (20.4)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
16 (42.1)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{0.004}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
12.0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
102 (100.0)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Table 1 represents the child and maternal characteristics for the study
population stratified for \emph{FLG} mutations. Values are based on the
non-imputed dataset and represented as number (\%). Chi-squared tests
were conducted to examine possible differences in baseline
characteristics between the different genotypes. Abbreviations:
\emph{FLG}, filaggrin gene; N, number; + Including 3 biallelic mutations
(2 compound heterozygous and 1 homozygous) \textsuperscript{++} Including
one biallelic mutations (homozygous) \selectlanguage{ngerman}§ Based on parental\selectlanguage{english}-reported
questionnaires obtained at the child's age of 10 years: ever atopic
dermatitis (no or yes)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Table 1 represents the child and maternal characteristics for the study
population stratified for \emph{FLG} mutations. Values are based on the
non-imputed dataset and represented as number (\%). Chi-squared tests
were conducted to examine possible differences in baseline
characteristics between the different genotypes. Abbreviations:
\emph{FLG}, filaggrin gene; N, number; + Including 3 biallelic mutations
(2 compound heterozygous and 1 homozygous) \textsuperscript{++} Including
one biallelic mutations (homozygous) \selectlanguage{ngerman}§ Based on parental\selectlanguage{english}-reported
questionnaires obtained at the child's age of 10 years: ever atopic
dermatitis (no or yes)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Table 1 represents the child and maternal characteristics for the study
population stratified for \emph{FLG} mutations. Values are based on the
non-imputed dataset and represented as number (\%). Chi-squared tests
were conducted to examine possible differences in baseline
characteristics between the different genotypes. Abbreviations:
\emph{FLG}, filaggrin gene; N, number; + Including 3 biallelic mutations
(2 compound heterozygous and 1 homozygous) \textsuperscript{++} Including
one biallelic mutations (homozygous) \selectlanguage{ngerman}§ Based on parental\selectlanguage{english}-reported
questionnaires obtained at the child's age of 10 years: ever atopic
dermatitis (no or yes)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Table 1 represents the child and maternal characteristics for the study
population stratified for \emph{FLG} mutations. Values are based on the
non-imputed dataset and represented as number (\%). Chi-squared tests
were conducted to examine possible differences in baseline
characteristics between the different genotypes. Abbreviations:
\emph{FLG}, filaggrin gene; N, number; + Including 3 biallelic mutations
(2 compound heterozygous and 1 homozygous) \textsuperscript{++} Including
one biallelic mutations (homozygous) \selectlanguage{ngerman}§ Based on parental\selectlanguage{english}-reported
questionnaires obtained at the child's age of 10 years: ever atopic
dermatitis (no or yes)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Table 1 represents the child and maternal characteristics for the study
population stratified for \emph{FLG} mutations. Values are based on the
non-imputed dataset and represented as number (\%). Chi-squared tests
were conducted to examine possible differences in baseline
characteristics between the different genotypes. Abbreviations:
\emph{FLG}, filaggrin gene; N, number; + Including 3 biallelic mutations
(2 compound heterozygous and 1 homozygous) \textsuperscript{++} Including
one biallelic mutations (homozygous) \selectlanguage{ngerman}§ Based on parental\selectlanguage{english}-reported
questionnaires obtained at the child's age of 10 years: ever atopic
dermatitis (no or yes)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Table 1 represents the child and maternal characteristics for the study
population stratified for \emph{FLG} mutations. Values are based on the
non-imputed dataset and represented as number (\%). Chi-squared tests
were conducted to examine possible differences in baseline
characteristics between the different genotypes. Abbreviations:
\emph{FLG}, filaggrin gene; N, number; + Including 3 biallelic mutations
(2 compound heterozygous and 1 homozygous) \textsuperscript{++} Including
one biallelic mutations (homozygous) \selectlanguage{ngerman}§ Based on parental\selectlanguage{english}-reported
questionnaires obtained at the child's age of 10 years: ever atopic
dermatitis (no or yes)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Table 1 represents the child and maternal characteristics for the study
population stratified for \emph{FLG} mutations. Values are based on the
non-imputed dataset and represented as number (\%). Chi-squared tests
were conducted to examine possible differences in baseline
characteristics between the different genotypes. Abbreviations:
\emph{FLG}, filaggrin gene; N, number; + Including 3 biallelic mutations
(2 compound heterozygous and 1 homozygous) \textsuperscript{++} Including
one biallelic mutations (homozygous) \selectlanguage{ngerman}§ Based on parental\selectlanguage{english}-reported
questionnaires obtained at the child's age of 10 years: ever atopic
dermatitis (no or yes)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}



\textbf{FIGURE LEGENDS}

\textbf{Figure 1. Flow chart of participants included in the study.}

Abbreviations: Th, helper T cell; Treg, regulatory T cell.; Tcm, central
memory T lymphocytes; TemRA, effector memory RA-positive T lymphocytes;
TemRO, effector memory RO-positive T lymphocytes

\textbf{Figure 2. Absolute numbers of blood T cell subsets stratified
by\emph{Filaggrin Gene} mutation status}

\emph{Figure 2A} shows the median (IQR) cell count per \selectlanguage{greek}μ\selectlanguage{english}Lblood for Th
and Treg cell numbers stratified for \emph{FLG} mutation. \emph{Figure
2B} shows the median (IQR) cell count per \selectlanguage{greek}μ\selectlanguage{english}Lblood for
CD4\textsuperscript{+} and CD8\textsuperscript{+} effector memory T cell
numbers stratified for \emph{FLG} mutation. \emph{Abbreviations} : IQR,
interquartile range; Tcm, central memory T lymphocytes; TemRA, effector
memory RA-positive T lymphocytes; TemRO, effector memory RO-positive T
lymphocytes; Th, helper T cell; Treg, regulatory T cell. * denotes a
two-sided P-value \textless{}0.05. Supplementary Table 1 and 3 show
absolute numbers and p-values.

\textbf{Figure 3. Absolute numbers of blood memory B cell subsets
stratified by \emph{Filaggrin Gene} mutation status} \emph{Figure
3}shows the median (IQR) cell count per \selectlanguage{greek}μ\selectlanguage{english}L blood for B memory cell
numbers stratified for \emph{FLG} mutation. Abbreviations: IQR,
interquartile range; Ig, Immunoglobulin. Supplementary Table 2 shows
absolute numbers and p-values.\selectlanguage{english}
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