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.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-offs3,6for 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
variables3 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.3
In a recently published genome-wide association study (GWAS)
investigating susceptibility to HPAS in asthmatic children, screening
was not performed for rs41423247 (GC).7Instead
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.8The 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.9Tsartsali et al5 and
others10reported 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.11The 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
Europe12) 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
(<2 x 10-16), which even far exceeds the
required significance threshold for a GWAS (5 x
10-8).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).14-16In
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.16More likely, it isjust a common variant
in the population (40% in our sample)presenting with reduced
glucocorticoid sensitivity.16
PGGR is associated with 26 mutations16and 8 pathogenic
SNPS17in the exome of NR3C1 at 5q31.3,while
rs41423247falls within the intronic regulatory region of the
gene,12confirmed 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
FEV1ofchildren with the GG genotype improved more after
a high dose of ICS than children with the CG+CC
genotype.18In the current study, three of five
children with the CC genotype had a suboptimal FEV1(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.1In 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 BMI11, in line with the
current study. However, others have found the
opposite.19Thehigher 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.
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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