Psychology, 2010, 1, 247-251
doi:10.4236/psych.2010.14033 Published Online October 2010 (http://www.SciRP.org/journal/psych)
247
Possible Genetic Dysregulation in Pediatric CFS*
Leonard A. Jason1, Matthew Sorenson1, Nicole Porter1, Molly Brown1, Athena Lerch1,
Constance Van der Eb1, Judy Mikovits2
1DePaul University, Chicago, USA; 2The Whittemore Peterson Institute for Neuroimmune Disease, Reno, Nevada
Received July 16th, 2010; revised July 26th, 2010; accepted July 28th, 2010.
ABSTRACT
Hypocortisolism is a frequent finding in individuals with chronic fatigue syndrome (CFS) and could play an explana-
tory role in the development of illness symptomatology. The etiologic mechanism behind this finding could be genetic
variance in glucocorticoid receptor expression (GR) or increased resistance to the effects of glucocorticoids. Several
investigators believe that allelic variance in a GR (NR3C1) mediates the expression of chronic fatigue possibly through
influence on hypothalamic-pituitary-adrenal (HPA) axis function [1]. In addition, several immunologic variables are
associated with CFS. The nuclear factor kappa beta (NFkB) pathway is heavily involved in cellular transcription and
regulation and has been shown to be associated with the development of CFS. The NFkB pathway is directly regulated
by and influences the presence of GR [2]. Our study focused on assessing whether such inflammatory transcription is
occurring during adolescent years. Findings indicated decreased expression of NFKB1, NFKB2, and NR3C1. A de-
crease in the expression of these genes may have effects on immune cell function and cytokine production that could
explain immunologic findings seen in individuals with CFS.
Keywords: Pediatric, Chronic Fatigue Syndrome, Hypocortisolism, Glucocorticoid Receptor Expression
1. Introduction
In adult populations, dysregulation of the hypotha-
lamic-pituitary-adrenal (HPA) axis has been associated
with CFS (Johnson & DeLuca, 2005) [3]. Adults with
CFS tend to display lower levels of cortisol, the main
signaling hormone of the HPA axis [4,5]. The literature
is replete with findings implicating HPA axis dysregula-
tion with CFS either through lower baseline cortisol [6],
a lack of responsiveness on the part of the HPA axis [7],
a pattern of glucocorticoid resistance [8], or disruption or
dysregulation of the expected diurnal cortisol pattern [9].
A gene for defective cortisol binding protein has been
associated with CFS [10]. Defective cortisol binding pro-
tein can lower the ability to respond to cortisol. Smith et
al. [11] found that increased expression of genes influ-
encing the HPA axis and changing cortisol production
predicted the prevalence of unexplained chronic fatigue.
A gene study by Rajeevan et al. [1] also implicated cor-
tisol in CFS, finding single nucleotide polymorphisms in
the glucocorticoid receptor (GR) gene (several such al-
leles were associated with increased risk for CFS). Cor-
tisol binding is influenced by elements of the neuroendo-
crine pathways.
Complex interactions of neurological, immune, and
endocrine systems, operating from the individual’s ge-
netic substrate, work in conjunction with environmental
factors to influence onset of CFS and its clinical course.
In particular, altered functioning of the HPA axis is im-
plicated in the reduced capability to regulate responses to
stress as seen in disorders such as CFS [12]. Variance in
the expression of genes associated with HPA axis func-
tion has been associated with CFS across several studies
with adult populations [13]. Previous work has found
variance in the expression of GR (NR3C1) in individuals
with CFS compared to controls [11]. Those with CFS
may have a decreased sensitivity to the effects of cortisol
due to a down-regulation of GR [8].
It is apparent that a degree of HPA axis dysfunction is
involved in the pathogenic process of adult CFS, and it is
possible that similar variables may predict the existence
of HPA axis dysfunction among pediatric CFS cases in-
volving children, adolescents, and young adults. For
example, Mathew et al. [14] found the presence of HPA
axis dysregulation in adolescence may serve as a predic-
tor of this illness. Miike et al. [15] found that cortisol
secretion was reduced in a pediatric population with CFS
*The authors appreciate the financial assistance provided by the National
Institute of Allergy and Infectious Diseases (grant number AI055735).

Possible Genetic Dysregulation in Pediatric CFS
248
in comparison to controls. Segal, Hindmarsh, and Viner
[16] found among adolescents with CFS a subtle altera-
tion in adrenal functioning suggesting a reduction in cen-
tral stimulation of adrenal glands, with females exhibit-
ing a more attenuated response to ACTH than males.
There is a consistent body of literature that has found
dysregulation of glucocorticoid function to be associated
with CFS in both adult and adolescent populations.
The present study focused on steroid receptor expres-
sion in a pediatric population with CFS, and we exam-
ined the presence of several specific genes associated
with the development of CFS in adult populations. One
of the primary hypotheses underlying the proposed work
is that a pattern of hypocortisolism seen in adults with
CFS [9] would be manifested in a pediatric population
with the same disease. In addition, NR3C1 is one of the
main transcriptional regulators of the GR, and polymor-
phisms of the NR3C1 gene have been associated with
CFS in adults [1]. We also measured NFKB1 and
NFKB2, which have been associated with inflammatory
responses known to be associated with the development
of CFS in adult populations [17]. The initiation of in-
flammatory changes might precede the development of
fatigue symptomatology by several years. Therefore, we
wanted to determine whether such inflammatory tran-
scription was occurring during adolescent years.
2. Method
Data were collected in the mornings from a sample of
adolescents with CFS (n = 6) which was obtained from
the Chicago metropolitan area. Five were Caucasian and
one Asian-American. Three were female and three were
male. The average age was 17.8 years (range from 16 to
21). All were diagnosed with CFS by a physician who
was familiar with this illness.
Serum cortisol served as an indicator of hypotha-
lamic-pituitary-adrenal (HPA) axis function. Serum was
obtained through centrifugation for a period of 20 min-
utes at 1000 X gravity. The collected samples were lab-
eled with a unique identifier and preserved in a –80 deg-
ree centigrade freezer until time of assay. A commerc-
ially available enzyme linked immunoabsorbent assay
(sensitivity: 0.030-3111 ng/ml) was used to determine
cortisol concentration (R&D Systems, Minneapolis, MN).
For the children, one tube of peripheral blood (10ml)
was preserved in lithium heparin. From this sample, pe-
ripheral blood mononuclear cells were obtained and pre-
served in accordance to a protocol provided by Panomics
Inc (Fremont, CA). Preserved cells were then shipped
to Panomics Inc. for analysis of the desired mRNA tran-
scripts using a QuantiGene Plex System. This system
employed blood lysate and branched DNA in creating a
sandwich nucleic acid hybridization, which was then
bound to a biotinalyted probe. Results were obtained
through the use of laser excited fluorescent signal. Sam-
ple mean fluorescent intensity was calculated in relation
to the mean signal of glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH). Sample plates were read through
the use of a BioPlex System (BioRad Laboratories, Her-
cules, CA).
Rationale for Selection of Cortisol and mRNA tran-
scripts:
We examined for the presence of messenger RNA
transcripts affecting receptor expression. The rationale
for the selection of the examined transcripts (NR3C1,
NFKB1, NFKB2) was based upon previous evidence
demonstrating a possible relationship between these tran-
scripts and CFS, along with an evaluation of their known
biologic activity. Based on previous findings with adult
populations, we also elected to examine cortisol level in
this population.
The NR3C1 gene encodes a protein receptor for glu-
cocorticoids. The encoded protein can bind to DNA and
other proteins influencing transcriptional regulation.
Mutations in the structure of this protein can influence
glucocorticoid binding resulting in a degree of resistance
to the actions of glucocorticoid.
The nuclear factor kappa beta (NFkB) pathway is one
of the main regulators of inflammatory response across
multiple cell populations. This pathway influences and is
influenced by the release of cytokines and other inflam-
matory mediators. NFKB1 is involved in cell differentia-
tion and pro-inflammatory immune response. NFKB2 is
also involved in cell differentiation and pro-inflammatory
immune response but has a stronger role on B cell lin-
ages and on apoptosis (programmed cell death). The ex-
pression of the nuclear receptor family NFKB1 (nuclear
factor of kappa light polypeptide, p105) and NFKB2
(nuclear factor of kappa light polypeptide gene enhancer
p 49/100) are also directly regulated by and influence the
presence of GR [18]. Determining the levels of NFKB1
and NFKB2 provides a means of determining relative
efficacy of GR function and an insight into regulation of
this receptor in an adolescent population with fatigue. In
this exploratory study, we chose to focus on those that
have been demonstrated in adult populations to be asso-
ciated with CFS. Thus, we examined for variance in the
expression of factors involved in the regulation and ex-
pression of discrete components of immune function in a
population of adolescents with CFS.
3. Results
The obtained samples demonstrated low mean cortisol
values (M = 56.73 ng/ml, SD = 24.73). These pediatric
CFS values are considerably lower than those found in
control pediatric samples (6-16 years of age, M = 91.0

Possible Genetic Dysregulation in Pediatric CFS
249
ng/ml, SD = 19) [19] and provide support for the pres-
ence of hypocortisolism in pediatric samples with CFS.
Additionally, the six samples were sent to the assay
service of Panomics (Fremont, CA) for determination of
three discrete genes conceptualized as associated with
development of CFS; NRC31, NFKB1 and NFKB2. Re-
sults were then normalized to GAPDH and expression
ratios derived. Mean expression ratios and ranges are
provided in Table 1.
With gene expression ratios, values less than one indi-
cate a down-regulation of function in relationship to the
housekeeping gene (GAPDH) whereas values greater
than one would indicate increased expression. These data
demonstrate a pattern of down-regulation of gene ex-
pression in the pediatric sample with CFS, concomitant
with reduced cortisol levels.
4. Discussion
The study’s main findings were hypocortisolism and the
down-regulated expression of NR3C1 (the encoding gene
for the GR), NFKB1, and NFKB2. The reduced expres-
sion of the gene for the GR provides evidence for dys-
function of the HPA axis in those with CFS. The expres-
sion of NFKB is associated with proinflammatory im-
munologic responses and is induced by stimuli such as
reactive oxygen species, mitogens, cytokines TNFα, and
IL-1. In our pediatric sample, there was a marked down-
regulation of NFKB1 and NFKB2. However, in response
to down regulated endogenous glucocorticoid levels,
transcription of inflammatory genes by NFKB might be
expected to be up regulated. Glucocorticoid has signifi-
cant suppressive effects on the expression of NFkB, an
action that occurs through ligand binding of the gluco-
corticoid receptor. In the presence of down-regulation of
the glucocorticoid receptor, it is possible that suppression
of NFkB is inactive. Alternatively, there may be a dis-
ruption of an associated co-receptor or molecule that
inhibits adequate GR binding, leading to a state in which
NFkb and NR3C1 levels are both reduced.
NFKB is a proinflammatory transcriptional factor and
prevents apoptosis and immune suppression which are
typically evidenced by up regulated CD8+. In a study of
girls with CFS, Ter Wolbeek et al., [20] also found de-
creased levels of the pro-inflammatory cytokines IL-6
and TNF-α, but found increased levels of the anti-in-
flammatory cytokines IL-10 and interferon (IF)-gamma.
However, evidence for a process of anti inflammatory
transcription or immune suppression would be supported
by a predominance of TH2 type immune response and
elevated cortisol levels [2].
One possible explanation for this down regulation of
all three genes is a bidirectional competitive inhibition or
trans repression exerted upon NFKB1/NFKB2 and
NR3C1 by one another. This process may be the end
result of a prolonged activation of the NFKB pathway,
such that expressions of NFKB1/NFKB2 are depleted via
the production of their own inhibitor IKBα upon translo-
cation to the nucleus. IKBα (a member of the IKB family
of inhibitory proteins) creates a feedback control loop
which prevents NFKB levels from becoming too high
and creating excessive inflammation. IKBα will inhibit
proinflammatory transcription by NFKB via nega- tive
feedback, which could possibly explain the down regula-
tion of the NFKB1and NFKB2 genes. The initiation of
the triggering of the NFKB pathway is likely dir- ected
by the presence of several factors: mitogens, bacte- ria,
UV exposure, viruses, cytokines IL-1 and TNF α along
with other proinflammatory, and reactive oxygen species
(all of which have the potential to trigger inflammation
and suppress cortisol levels) [18,21]. It is possible that a
prolonged activation of this inflammatory pathway has
led to the down regulation of the production of NFKB1
and NFKB2 by its inhibitor IKBα. This inhibition would
then leave the cell in an anti inflammatory state, possibly
leading to the down regulation of the NR3C1 gene cod-
ing for the GCR, as its expression would not be neces-
sary when the cell is in an anti inflammatory or sup-
pressed state.
A second possible explanation for why all three genes
are down regulated is competition between NFKB and
the GCR for limited amounts of the following co activa-
tors: steroid receptor coactivator-1 (SRC-1) and CREB-
Binding protein (CBP). Both of these co activators bind
to NR3C1 and NFKB and are necessary for the transac-
tivation (transport into the nucleus where transcription is
carried out) of both NFKB and the GCR [22]. Inadequate
supplies or insufficient levels of these co activators could
lead to the down regulated expression of each of the
three genes that code for transcriptional factors. It is also
possible that NFKB1 and NFKB2 are involved in affect-
ing the degree to which the glucocorticoid receptor (GCR)
displays sensitivity to its ligand (cortisol). Both the ex-
pression and sensitivity of the GCR to its ligand may be
affected by any number of factors including but not
Table 1. Mean normalized expression ratios and ranges for genes of interest.
Variable NFkB1 NFkB2 NR3C1
Pediatric 0.09 (0.05-0.13) 0.10 (0.04-0.14) 0.04 (0.02-0.10)
Universal RNA 0.23 0.41 0.65

Possible Genetic Dysregulation in Pediatric CFS
250
limited to: posttranslational modifications, the effects of
signaling cascades, reduced or altered expression of heat
shock proteins, DNA bending, variations in the receptor
protein, dimerization of an alternative receptor, recaptor
chaperone defect [18,21].
These data demonstrate that in a pediatric population
with CFS, there is decreased expression of the gene en-
coding for the GR. In an adolescent population, there
may be an increased level of vulnerability to disruptions
of the HPA axis. In a vulnerable population, such disrup-
tions may have end effects on cognitive pathways and
lifelong neuroendocrine responsiveness [23]. While
much of the speculation of the effects of cortisol on de-
velopment has tended to investigate the effects of hyper-
cortisolism, the lack of adequate hormone may be as
equally disruptive. These data highlight the importance
of examining this pathway in an adolescent population.
These findings should be considered as preliminary,
given the small sample size, and there is a need for rep-
lication with a larger data set.
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