Overexpression of the Per2 Gene in Male Patients with Acute Q Fever
Service d'Anesthsie et de Ranimation
, Hpital Nord, Assistance Publique-Hpitaux de Marseille,
Received 13 March 2012; accepted 28 June 2012; electronically published 14 September 2012. Nord, Chemin des Bourrely,
13915 Marseille, France
Unit de Recherche sur les Maladies Infectieuses Tropicales et Emergentes, Aix-Marseille Universit
The prevalence of Q fever is higher in men than in women. Because the expression of circadian clock genes differs in male and female mice infected with Coxiella burnetii, we hypothesized that circadian genes are differently modulated in men and women with Q fever. The expression of the Per2 gene was significantly (P = .01) increased in males with acute Q fever compared with healthy volunteers. No significant difference was observed in females. We showed for the first time that gender altered the expression of a circadian gene, Per2, in an infectious disease.
The study was conducted with the approval of the Ethics
Committee of the Aix-Marseille University, Marseille, France.
Informed written consent was obtained from each participant.
Blood samples from patients undergoing infectious disease
consultations and healthy volunteers were collected in
PAXgene Blood RNA tubes (Qiagen, Courtaboeuf, France).
The samples were collected from 9:00 am to 11:00 am. The
diagnosis of Q fever was performed as reported previously .
Q fever patients were split into Acute and Endocarditis
according to their underlying disease . The gene expression
in blood samples was determined with real-time quantitative
reverse transcriptase polymerase chain reaction (qRT-PCR) as
recently described . In brief, total RNA was extracted after
DNase digestion, according to the manufacturers
recommendations (Invitrogen, Life Technologies, Saint-Aubin, France).
Real-time PCR with cDNA templates was performed using
Light Cycler-FastStart DNA MasterPLUS SYBR Green I (Roche
Diagnostics, Basel, Switzerland). The primers were designed
with the free Web software Primer3 (http://frodo.wi.mit.edu/).
The primers consisted of the forward sequences 5-CGGAGT
GAGAGG-3 and 5-CCCTCTACCTGCTCAAA-GAAAA-3
and the reverse sequences 5-GGGACTGGAAA-ATGCT
GAGTT-3, 5-AAGGATA-ACAGCAAAGCAGAGG-3 and
5-GCCCTCTGGTCT-ACAAAAACAA-3 for the Per2, Clock,
and Arntl genes, respectively. Quantitative RT-PCR experiments
were performed using the gene encoding -actin as the
reference housekeeping gene. For each patient, the cycle
threshold (CT) values of the genes of interest were normalized
with the patients CT values of -actin to calculate the CT.
The results are expressed as the median and were compared
using the nonparametric MannWhitney U test. A P value
less than .05 was considered significant.
Our cohort consisted of 14 men (mean age of 51 4 years),
including 9 patients with acute Q fever and 5 with Q fever
related endocarditis; 6 women (mean age of 35 3 years),
including 5 patients with acute Q fever and 1 with Q
feverrelated endocarditis; and 12 healthy volunteers,
including 6 men (mean age of 41 4 years) and 6 women (mean age
of 39 5 years). With respect to gender, age did not differ
significantly between the patients and the healthy volunteers. In
contrast, among the patients, the men were older than the
women (51 4 vs 35 3 years; P = .02). No difference was
noted between men and women in the healthy volunteers.
In men, the expression of the Per2 gene was significantly
higher in the patients with acute Q fever than in the healthy
volunteers (FC = 3.1; P = .01). No significant difference of
expression was observed between Q feverrelated endocarditis
and healthy controls (P = .18) (Figure 1A). In women, the Per2
expression ratio between acute Q fever and healthy controls did
not reach a significant level (FC = 1.8; P = .18) (Figure 1B). In
contrast to the expression of the Per2 gene, the expression of
the Clock and Arntl genes was not affected in men or women
with Q fever compared with healthy volunteers (Figure 1CF).
To our knowledge, we showed significant changes in circadian
clockrelated gene expression in a human infectious disease
for the first time. Indeed, the expression of the Per2 gene was
increased in men but not in women with acute Q fever. This
result confirmed that C. burnetii infection partly affects the
circadian clock, as demonstrated in mice . This effect was
independent of the time of blood collection because blood was
sampled within a 2-hour period.
The human infection shows striking differences compared
with the murine model of C. burnetii infection. First, the
expression of the Per2 gene is upregulated in female mice ,
but it is obvious that the circadian clock is inverted in rodents
and humans . Second, the expression of the Arntl and
Clock genes is modulated in female mice infected with C.
burnetii. However, this finding was not confirmed in the present
study. These differences may be induced by specificities at the
tissue level. Indeed, the expression of the Per2, Arntl, and
Clock genes was studied in the liver of mice, whereas blood
samples were collected in humans. Note that significant
differences have been reported in the expression of the Clock
gene between peripheral tissues and blood .
In humans, the exposure to C. burnetii is similar for both
genders, although the prevalence of Q fever is higher in men
than in women . This explains the small number of women
included in our study. Murine models have shown that the
bacterial load is higher in the spleen of males than in females
. In Drosophila, phagocytosis is circadian-regulated by the
protein Timeless. This protein appears to modulate an
upstream event in phagocytosis . Because the survival of
C. burnetii in macrophages is mediated by a subversion of
receptor-mediated phagocytosis , this result suggests that the
impaired bacterial clearance in males may be related to these
circadian clock alterations [2, 3]. Future studies are required to
test this hypothesis.
Interestingly, the expression of the Per2 gene was increased in
men with acute Q fever but not in those with C. burnetii
endocarditis, suggesting that the expression of the Per2 gene is related
to the clinical status of the patient. It has been demonstrated that
the immune response of patients with acute Q fever is largely
different from that of patients with C. burnetii endocarditis .
Among the factors affecting the chronic evolution of Q fever,
aging may be essential. Indeed, in mice, aging is associated with
a reduced amplitude of circadian clock relatedgene expression
. Age affects both the expression of circadian clockrelated
genes and the response of mice to C. burnetii infection . In
our cohort, the female patients were younger than the male
patients. Future studies are required to identify the correlation
between age and gender in the expression of the Per2 gene.
In conclusion, we reported for the first time that the
expression of the Per2 gene was increased in men with acute Q fever
but not in women. This gender dimorphism may be related to
the higher incidence and severity of Q fever in men than in
women. This finding provides new perspectives to investigate
the pathophysiology of Q fever.
Acknowledgments. Vikram Mehraj is supported by the Higher
Education Commission (Pakistan) and the Assistance PubliqueHpitaux de
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.