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\begin{document}

\title{Hypothalamic-pituitary-adrenal axis suppression in asthma: A
glucocorticoid receptor polymorphism may protect}



\author[1]{Wisdom Akurugu}%
\author[2]{Carel van Heerden}%
\author[1]{Nicola Mulder}%
\author[2]{Ekkehard Zöllner}%
\affil[1]{University of Cape Town Faculty of Health Sciences}%
\affil[2]{Stellenbosch University}%


\vspace{-1em}



  
  \date{June 12, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Asthmatic children on corticosteroids can develop
hypothalamic-pituitary-adrenal axis suppression (HPAS). Single
nucleotide polymorphisms (SNPs) rs242941 and rs1876828 of the
corticotrophin-releasing hormone receptor 1 (CRHR1) gene were associated
with lower stimulated cortisol (F) levels, whereas rs41423247 of the
glucocorticoid receptor (NR3C1) gene was associated with higher basal F
levels. The objective of the current study was to confirm whether these
three SNPs are associated with HPAS in asthmatic children. Methods: DNA
was extracted from saliva obtained from 95 asthmatic children, who had
previously undergone basal F and metyrapone testing. Thirty-six children
were classified as suppressed. Non-suppressed children were
sub-classified according to their post-metyrapone ACTH (PMTP ACTH) level
into a middle (106-319 pg/ml) and a high (\textgreater{}319 pg/ml) ACTH
response group. TaqMan\selectlanguage{ngerman}® polymerase chain reaction assays were utilized.
Results: Only rs41423247 was inversely associated with HPAS (OR = 0.27
{[}95\% CI 0.06-0.90{]}). Its GC genotype was inversely associated with
HPAS (log odds = - 1.28, p = 0.021). \selectlanguage{english}[?]PMTP ACTH was associated with CC
(effect size = 10.85, p = 0.005) and GC genotypes (effect size = 4.06, p
= 0.023). The C allele is inherited as a dominant trait (effect size =
-1.31 (95\% CI -2.39 -- -0.33; p = 0.012). In the high ACTH response
group, both genotypes affected the PMTP ACTH (effect sizes 1.41 and
15.46; p-values 0.023 and \textless{} 2x10-26 for GC and CC
respectively). Conclusions: The C allele of rs41423247 was found to be
protective against HPAS. CC genotype is associated with the highest PMTP
ACTH response.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Hypothalamic-pituitary-adrenal axis suppression in asthma: A
glucocorticoid receptor polymorphism may protect}

Wisdom Alemya Akurugu*\textsuperscript{1)}

CarelJacobusvan Heerden\textsuperscript{2)}

Nicola Mulder\textsuperscript{1)}

Ekkehard Werner Z\selectlanguage{ngerman}öllner*\textsuperscript{3)}

* WAA and EWZ should be considered joint first author

\textbf{Institutional affiliations}

\textsuperscript{1)}Computational Biology, Department of Integrative
Biomedical Sciences, University of Cape Town, Cape Town, South Africa

\textsuperscript{2)}Central Analytical Facility, Stellenbosch
University, Stellenbosch, South Africa

\textsuperscript{3)}Paediatric Endocrine \& Diabetes Unit, Department of
Paediatics and Child Health, Stellenbosch University, Cape Town, South
Africa

\textbf{Running title} : HPAS, GC receptor polymorphism in asthma

\textbf{Author responsible for correspondence}

EW Zöllner

Faculty of Health Sciences

Department of Paediatrics and Child Health

Stellenbosch University

PO Box 241

Cape Town

8000

South Africa

\textbf{Word count} : 2480

\textbf{Word count abstract:} 250

\textbf{Title character count} : 99

\textbf{No of tables:} 5

\textbf{No of figures:} 1

\textbf{Conflict of interest}

WAA, CJVH, NM \& EWZ have nothing to disclose.

\textbf{Financial support}

This study was supported by grants from the National Research Foundation
and the South African Thoracic Society.

\textbf{Abstract and keywords}

\textbf{Background:} Asthmatic children on corticosteroids can develop
hypothalamic-pituitary-adrenal axis suppression (HPAS). Single
nucleotide polymorphisms (SNPs) rs242941 and rs1876828 of the
corticotrophin-releasing hormone receptor 1 (CRHR1) genewere associated
with lower stimulated cortisol (F) levels,whereas rs41423247 of the
glucocorticoid receptor (NR3C1) genewas associated with higher basal F
levels. The objective of the current study was to confirmwhether these
three SNPs are associated with HPAS in asthmatic children.

\textbf{Methods:} DNA was extracted from saliva obtained from
95asthmatic children, who had previously undergone basal F and
metyrapone testing. Thirty-six children were classified as suppressed.
Non-suppressed children were sub-classified according to their
post-metyrapone ACTH (PMTP ACTH)level into a middle (106-319 pg/ml) and
a high (\textgreater{}319 pg/ml) ACTH response group.
TaqMan\textsuperscript{®}polymerase chain reaction assays were utilized.

\textbf{Results:} Only rs41423247 was inversely associated with HPAS (OR
= 0.27 {[}95\% CI 0.06-0.90{]}). Its GC genotype was inversely
associated with HPAS (log odds = - 1.28, p = 0.021). \selectlanguage{english}[?]PMTP ACTH was
associated with CC (effect size = 10.85, p = 0.005) and GC genotypes
(effect size = 4.06, p = 0.023). The C allele is inherited as a dominant
trait (effect size = -1.31 (95\% CI -2.39 -- -0.33; p = 0.012). In the
high ACTH response group, both genotypes affected the PMTP ACTH (effect
sizes 1.41 and 15.46; p-values 0.023 and \textless{}
2x10\textsuperscript{-26} for GC and CC respectively).

\textbf{Conclusions:} TheC allele of rs41423247was found to be
protective against HPAS. CC genotype is associated with the highest
PMTPACTH response.

\textbf{Key words:} Asthma, hypothalamus, pituitary, adrenal,
polymorphism, receptors, glucocorticoids, steroids/adverse events,
adrenal insufficiency, pituitary-adrenal-function tests

\textbf{Main text}

\textbf{Key Messages}

Rs41423247 (G/C) of the NR3C1 gene was found to be inversely associated
with hypothalamic-pituitary-adrenal axis suppression (HPAS) in asthmatic
children on steroids. The CC genotype is associated with the highest
ACTH levels. If confirmed, the CC genotype could well be a marker of
HPAS protection, thereby allowing for personalized treatment decisions
to be made in asthma care.

\textbf{1 BACKGROUND}

It has been shown that 16.3 \% of asthmatic children treated with
inhaled corticosteroids (ICS) and nasal steroids (NS) in Cape Town,
South Africa, can develop hypothalamic-pituitary-adrenal axis
suppression (HPAS) when assessed with the metyrapone(MTP)
test.\textsuperscript{1-2} If partial forms of suppression are included,
even two-thirds of children may be affected. A higher body mass index
(BMI) was found to be protective against HPAS. Unfortunately suitable
screening tests for HPAS are not available.\textsuperscript{3-4}The only
viable alternative thus would be to identify gene variants, which would
help to predict which children are either prone or resistant to
developing HPAS. In Greek asthmatic childrenon inhaled
corticosteroids,homozygotes for the single nucleotide polymorphisms
(SNPs) rs242941 (TT) and rs1876828 (AA) of the corticotrophin-releasing
hormone receptor 1 (CRHR1) gene were associated with lower stimulated
cortisol (F) levels when tested with the low dose adrenocorticotropin
(ACTH) stimulation test (0.5\selectlanguage{greek}µ\selectlanguage{english}g/1.73m\textsuperscript{2}). Heterozygotes
(GC) for rs41423247 (\emph{Bcl} l restriction length polymorphism) of
the glucocorticoid receptor (NR3C1) gene,on the other hand,were found to
have higher basal F levels.\textsuperscript{5} In the quest for
universally applicable predictors of HPAS in asthmatic children on
corticosteroids, the findings of the Greek study needed to be confirmed
by a study, utilizing the gold standard metyrapone test, to determine
whether the SNPs rs242941 and rs1876828 of the CRHR1, and rs41423247 of
the NR3C1 gene are associated with HPAS in asthmatic school children on
corticosteroids.

\textbf{2 METHODS}

2.1 \textbf{Participants}

Asthmatic children, 5-18 years old, treated with ICS and NS at the Lung
Institute, Tygerberg and Red Cross Children's Hospitals in Cape Town,
South Africa, whose hypothalamic-pituitary-adrenal axis (HPA) was
previously assessed with a morning basal serum F and MTP testing,were
re-recruited.\textsuperscript{1-2}HPAS was diagnosed, if F\textless{} 83
nmol/l or the post-MTP (PMTP) ACTH \textless{} 106 pg/ml (23.5 pmol/l),
11-deoxycortisol (11DOC) \textless{} 208 nmol/l and 11DOC+C \textless{}
400 nmol/l.

2.2 \textbf{Study design and methodology}

A cross-sectional study was performed. Height, weight, sex and age were
recorded as measured previously. The BMI z-score (Centre of Disease
Control) was computed. Salivary samples were collected with an Oragene
DNA collection kit (OG-500). Samples were stored at - 20\selectlanguage{ngerman}º C. Once all
the samples were collected, DNA was extracted. Genotyping for rs242941,
rs1876828 and rs41423247 was performed by using
TaqMan\textsuperscript{®} polymerase chain reaction (PCR) assays.Ethical
approval was granted by the ethics committees of both Stellenbosch
University and the University of Cape Town. The study conformed to the
standards of the Declaration of Helsinki. All participants and their
parents gave their informed consent prior to inclusion of the study.

2.3 \textbf{Statistical analysis and sample size considerations}

2.3.1 \textbf{Sample size}

In the previous study\textsuperscript{1}30 patients had
hypocortisolaemia or HPAS. The cross-sectional design allowed for an
equal number of patients to be analysed across the full spectrum of
post-MTP responses i.e. low, midrange and high (30 subjects each). The
ACTH mean and standard deviation (SD) were calculated (ACTH
mean=330pg/ml {[}73.3 pmol/l{]}, SD=280pg/ml {[}62.2 pmol/l{]}) from the
previous data. The proportional distribution of the genetic subgroups
was derived from the paper of Tsartsali et al, table
4.7.\textsuperscript{5}Power analysis with a sample size of 90 was done
across the three ACTH groups. Significance level was taken as 0.05. A
one-way analysis of variance test (ANOVA)was considered for the power
analysis. The following mean values were specified under the alternative
hypothesis: group 1 (low range) 230pg/ml (51.1 pmol/l), group 2
(midrange) 330pg/ml (73.3 pmol/l), group 3 (high range) 430pg/ml (95.5
pmol/l). This gives a 100 ACTH units difference between two adjacent
groups. This is a relative difference of 36\% of the SD. {[}(100/280)=
0.36{]} which is a conservative expected effect size. Even for
unbalanced groups such as n1=24, n2=35 and n3=40, the power will be
81\%. Thus the power is good if the sample size distribution across the
genetic subgroups is moderately unbalanced. Power is improved by square
root transformations of ACTH to stabilise the within group variances.

2.3.2 \textbf{Statistical analysis}

An online statistical tool
(https://ihg.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl) and the R
statistical packagewere used. Fisher's Exact and Pearson's
Chi-squaretestswere conducted to test for statistical difference of
minor allele counts. The\emph{HardyWeinberg} package version 1.6.3
installed in RStudio version 3.5.1 was used to assess whether the SNPs
were inHardy-Weinberg equilibrium. To test for significance at SNP and
genotype level, the Wald's test in \emph{multinom()} function from
the\emph{nnet} package in R was utilized. PMTP ACTH data were square
root transformed and a one-way ANOVA performed. The Wilcoxon test was
used for pairwise multiple comparisons of differences in mean \selectlanguage{english}[?]PMTP ACTH
per SNP. Linear analysis for BMI z-score, \selectlanguage{english}[?]PMTP ACTH and binomial
analysis for HPAS were conducted with the \emph{glm()} function in R.
The best genetic model for rs41423247 was selected on the basis of the
lowest significant Akaike information criterion. Significance level for
all tests was taken as 0.05.

3 \textbf{RESULTS}

A total of 96 patients (94 from previous studies\textsuperscript{1,2}and
two newpatients) were recruited. One child had to be excluded, becauseof
a discrepancy between the basal F level and the MTP test. The
demographics, therapy and HPAS status of the 95 enrolled patients
arelisted (table 1).Only one child was on oral prednisone at the time of
testing. According to the basal F and the PMTP ACTH response, 35
children were classified in asuppressed (F \textless{} 83 nmol/l or ACTH
\textless{} 106 pg/ml {[}23.5 pmol/l{]}), 29 in a middle (ACTH 106-319
pg/ml {[}23.5-70.8 pmol/l{]}) and 31 in a high (ACTH \textgreater{} 319
pg/ml {[}70.8 pmol/l{]}) ACTH response group.

All three SNPs were in Hardy-Weinberg equilibrium (rs242941A/C
{[}suppressed ACTH response group p=0.830;middle/high ACTH response
group p=0.580{]}, rs1876828 A/T {[}suppressed ACTH response group
p=0.791;middle/high ACTH response group p=0.481{]} and rs41423247G/C
{[}suppressed ACTH response group p=0.445;middle/high ACTH response
group; p=0.948{]}).

The rs41423247 (G/C) SNP was inversely associated with HPAS (OR = 0.27
{[}95\% CI 0.06-0.90{]}). Both rs242941 (A/C) and rs1876828 (C/T) were
not associated with HPAS, their OR being 0.99 (95\% CI 0.43-2.27) and
0.54 (95\% CI 0.05-3.48) respectively. Similarly, only the rs41423247 GC
and GC+CC genotypes were inversely associated with the suppressed ACTH
response group(OR=0.310 {[}95\% CI: 0.137-0.920{]} and OR = 0.296
{[}95\% CI: 0.1116 - 0.756{]}). The homozygous CC genotype just missed
statistical significance (OR = 0.101 {[}95\% CI: 0.005-1.908{]}, p =
0.041). The mean \selectlanguage{english}[?]PMTP ACTH of the CC genotype was significantly higher
(p = 0.002) than for the GC and GG genotypes (fig 1). The respective
means and their 95\% CIs were 22.59 (16.18-29.00), 16.00 (13.00-19.00)
and 11.82 (10.11-13.63). These correspond to real ACTH mean levels of
553.0 pg/ml (122.9 pmol/l) for the CC, 331.0 pg/ml (73.6 pmol/l) for the
GC and 187.5 pg/ml (41.7 pmol/l) for the GG genotype.

The frequency of the guanosine (G) allele (the major allele) of
rs41423247 was 77\%, while the frequency of the cytidine (C)allele (the
minor allele) was 23\%. The genotype frequencies for GG, GC and CC were
60, 34 and 6\% respectively.

BMI was associated with the CC genotype (table 2) while the heterozygous
GC was associated with HPAS, independently of BMI (table 3). Both the
homozygous CC and the heterozygous GC genotypes were associated with
\selectlanguage{english}[?]PMTP ACTH with CC having a significant larger impact than GC (table 4).
The observed ACTH effect was independent of age, sex, height and weight
(and hence of BMI). The higher the ACTH response, the greater and more
significant the genotype (CC) effect (table 5).

After correcting for age, sex, height and weight, both the additive and
the dominant genetic models were the best fit to describe the protective
effect of the C alleleon HPAS. The effect size for the additive model
was -1.30 (-2.32 -- -0.42; p = 0.007) and for the dominant model, the
effect size was -1.31 (95\% CI -2.39 -- -0.33; p = 0.012). Similarly,
the effect of the C allele on \selectlanguage{english}[?]PMTP ACTH is best described by an
additive (effect size = 4.42 {[}95\% CI 1.77 -- 7.07; p = 0.002{]}) and
a dominant model (effect size = 4.79 {[}95\% CI 1.54-8.04; p = 0.005).

\textbf{4 DISCUSSION}

The SNP rs41423247 (GC) of theNR3C1 gene was found to be protective
against HPAS, while no association with HPAS was found with rs242941 and
rs1876828 of the CRHR1 gene. In contrast, in Greek asthmatic
childrenbasal or stimulated F levels were differentfor all three
SNPs.\textsuperscript{5} Thediscrepancy in the findings between results
of the two studies can be explained by the use of different adrenal
function tests. In the Greek study the low dose ACTH stimulation test
(not the gold standard) was used without defining definitive
cut-offs\textsuperscript{3,6}for basal and stimulated F levels. Relying
on statistical differences of these levels vis-a-vis their respective
genotypes could have led to erroneous conclusions. Furthermore, serum F
is a very crude test, which is influenced by many
variables\textsuperscript{3} e.g. by Circadian rhythm. There is no
record in Tsatsali et al's paper whether the basal F levels were taken
in the early morning (which is essential). The MTP test, utilized in
this study, is a gold standard adrenal function test with clearly
defined criteria for HPAS. Analyses based on this test can be expected
to produce valid results.\textsuperscript{3}

In a recently published genome-wide association study (GWAS)
investigating susceptibility to HPAS in asthmatic children, screening
was not performed for rs41423247 (GC).\textsuperscript{7}Instead
rs591118, an intronic variant of the PDGFD gene, was significantly
associated with HPAS. This particular SNP was not included in the
current study, because the study was already completed when the paper
reporting rs591118 was published.

In the current study, CC of rs41423247 is associated with higher ACTH
levels when compared to theGCand GG genotypes. In adults with depression
on the other hand, a dexamethasone/corticotropin-releasing hormone (CRH)
test revealed that a poor response to anti-depressive therapy was
associated with marginally lower median peak ACTH levels in patients
with the CC genotype.\textsuperscript{8}The results of this study could
however have been confounded by the use of a variable dose of
triiodothyronine given to some patients,which could have
inducedunrecognised adrenal insufficiency in individuals at
risk.\textsuperscript{9}Tsartsali et al\textsuperscript{5} and
others\textsuperscript{10}reported that the GC genotype was associated
with a significantly higher basal F level than the CC genotype. The GG
genotype converselywas associated with lower F levels after a
dexamethasone suppression test in Dutch
elderlyindividuals.\textsuperscript{11}The authors interpreted this as
``hypersensitivity'' to glucocorticoids. However, on re-analysis of
their paper, CC is associated with a significantly higher F level
post-dexamethasone, suggesting glucocorticoid resistanceof the CC
genotype.

Both the HPAS-protective effect and the ACTH response seem to be
inherited in an additive and dominant fashion. A similar phenotype is
therefore expected withthe heterozygous GCand homozygous CCgenotype,
except that the phenotypic effect of the latter should be more
pronounced. Although the overall sample size was probably adequate, the
low minor allele frequency of 23\% (compared to 38\% in
Europe\textsuperscript{12}) and the low CC genotype frequency of 6\%
(compared to 40\% in Greek children), limit the interpretation of the
study. This resulted in overlapping CIs of the GC and CC genotypesand an
incalculable lower bound of the CC CI.However, the additive genetic
effectaccounted for a huge effect size and a highly significant p-value
(\textless{}2 x 10\textsuperscript{-16}), which even far exceeds the
required significance threshold for a GWAS (5 x
10\textsuperscript{-8}).\textsuperscript{13} Hence, pending confirmation
in a larger study, the most suitable marker for HPAS protection would
still be rs41423247(CC) of theNR3C1 gene.

The biochemical phenotype attributed to both CC and GC genotypes
ofrs41423247is akin to the one described in the syndrome of primary
generalized glucocorticoid resistance (PGGR).\textsuperscript{14-16}In
this syndrome there is dysregulation of F feedback at hypothalamic and
pituitary level, resulting in elevated ACTH and possibly F levels. In
the current study, early morning basal F levels of the children with
rs41423247were not elevated (results not shown).Clinical features of
this syndrome e.g. hypertension, hirsutism or premature pubarche,were
not detected.Hence, the biochemical phenotype of rs41423247(GC and CC)
couldrepresent tissue-specific glucocorticoid
resistance.\textsuperscript{16}More likely, it isjust a common variant
in the population (40\% in our sample)presenting with reduced
glucocorticoid sensitivity.\textsuperscript{16}

PGGR is associated with 26 mutations\textsuperscript{16}and 8 pathogenic
SNPS\textsuperscript{17}in the exome of NR3C1 at 5q31.3,while
rs41423247falls within the intronic regulatory region of the
gene,\textsuperscript{12}confirmed independently by variant browser
analysis(result not shown). Mechanistically, impaired promoter function
would lead to impaired DNA transcription, RNA translation and protein
production.

In a recently published study on asthma children in Turkey, the
FEV\textsubscript{1}ofchildren with the GG genotype improved more after
a high dose of ICS than children with the CG+CC
genotype.\textsuperscript{18}In the current study, three of five
children with the CC genotype had a suboptimal
FEV\textsubscript{1}(result not shown). Unfortunately the number is too
small to draw any valid conclusions from it.

In the same study population, it was previously shown that, as the BMI
z-score rises, the ACTH level rises.\textsuperscript{1}In the current
study, an association between CC genotypeand BMI z-score could be
demonstrated. No association of genotype was found with overweight or
obesity (results not shown). In Dutch elderly the GG genotype was
associated with a lower BMI\textsuperscript{11}, in line with the
current study. However, others have found the
opposite.\textsuperscript{19}Thehigher BMI z-score associated with
rs41423247is probably mediated through periodically elevated F levels.
However, as shown, the BMI z-score does not confound the SNP effect on
the ACTH level.

As findings in Greek and South African children (and possibly Turkish
children and Dutch adults) are similar, it is likely that the results of
this study are generalizable across all populations and ages.
Pathophysiological and molecular explanations of the observed phenotype
should also be equally applicable to all humans. However,
inadvertentlythe study islimited by the small sample of children with
the CC genotype. Hence, alarger study should be performed to confirm the
finding. In addition, a ``proof of concept'' study (screening asthmatic
children for rs41423247 (CC), and when positive, testing them with the
MTP testto confirm the phenotype)should be done. If confirmed,
rs41423247 (CC), as a marker of HPAS protection, should be screened for
in asthmatic children on glucocorticoids. This would allow clinicians to
choose which children can be safely treated with higher doses of
glucocorticoids -- afirst step towards personalized medicine in asthma
therapy.

In conclusion, rs41423247(GC) of theNR3C1 gene was found to be
protective against HPAS. The CC genotype was associated with the highest
ACTH levels. TheC allele is inherited as an additive and dominant trait.
The GCgenotype was independently associated with BMI z-score. If
confirmed by alarger and a ``proof of concept'' study,rs41423247 (CC)
could well be a marker of HPAS protection, thereby allowing for
personalized treatment decisions to be madein asthma care.

\textbf{ACKNOWLEDGEMENTS}

We are grateful to Carl Lombard from the Medical Research Council for
calculating the sample size. Sisters Poggenpoel and Van Wyk are thanked
for collecting the salivary samples of the patients. We express our
gratitude toAlvera Vorsterfrom the Central Analytical Facility for the
DNA extractions and genotyping of the participants. We thank Maia
Lesosky from the School of Public Health, University of Cape Town, for
statistical advice. For their advice on genetic modelling we are
indebted to Heather Cordell from the International Centre for Life,
Newcastle University and Ahmed Rabie, Centre of Biotechnology of Sfax,
Tunisia.

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\end{enumerate}

\textbf{Hosted file}

\verb`HPAS intron publication_tables.docx`  available at \url{https://authorea.com/users/323697/articles/452248-hypothalamic-pituitary-adrenal-axis-suppression-in-asthma-a-glucocorticoid-receptor-polymorphism-may-protect}

\textbf{Hosted file}

\verb`HPAS intron publication_figure.docx`  available at \url{https://authorea.com/users/323697/articles/452248-hypothalamic-pituitary-adrenal-axis-suppression-in-asthma-a-glucocorticoid-receptor-polymorphism-may-protect}

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