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Leptin promoter methylation in female patients with painful multisomatoform disorder and chronic widespread pain

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Background Different functional somatic syndromes (FSS), fibromyalgia (FMS) and other unexplained painful conditions share many common clinical traits and are characterized by troubling and functionally disabling somatic symptoms. Chronic pain is most frequently reported and at the center of patients’ level of disease burden. The construct of multisomatoform disorder (MSD) allows to subsume severely impaired patients suffering from FSS, FMS and other unexplained painful conditions to be examined for common underlying processes. Altered leptin levels and a pathological response of the HPA-axis as a result of chronic stress and childhood trauma have been suggested as one of the driving factors of disease development and severity. Previous studies have demonstrated that methylation of the leptin promoter can play a regulatory role in addiction. In this study, we hypothesized that methylation of the leptin promoter is influenced by the degree of childhood traumatization and differs between patients with MSD and controls. A cohort of 151 patients with MSD and 149 matched healthy volunteers were evaluated using clinical and psychometric assessment while methylation level analysis of the leptin promoter was performed using DNA isolated from whole blood. Results In female controls, we found CpG C-167 to be negatively correlated with leptin levels, whereas in female patients CpG C-289, C-255, C-193, C-167 and methylation cluster (C-291 to C-167) at putative bindings sites for transcription factors Sp1 and c/EBPalpha were negatively correlated with leptin levels. Methylation levels were significantly lower in female patients CpG C-289 compared with controls. When looking at female patients with chronic widespread pain methylation levels were significantly lower at CpG C-289, C-255 and methylation cluster (C-291 to C-167). Conclusion Our findings support the hypothesis that epigenetic regulation of leptin plays a role in the regulation of leptin levels in patients with MSD. This effect is more pronounced in patients with chronic widespread pain.
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Achenbachetal. Clinical Epigenetics (2022) 14:13
https://doi.org/10.1186/s13148-022-01235-5
RESEARCH
Leptin promoter methylation infemale
patients withpainful multisomatoform disorder
andchronic widespread pain
Johannes Achenbach1* , Mathias Rhein3, Alexander Glahn3, Helge Frieling3 and Matthias Karst2
Abstract
Background: Different functional somatic syndromes (FSS), fibromyalgia (FMS) and other unexplained painful
conditions share many common clinical traits and are characterized by troubling and functionally disabling somatic
symptoms. Chronic pain is most frequently reported and at the center of patients’ level of disease burden. The con-
struct of multisomatoform disorder (MSD) allows to subsume severely impaired patients suffering from FSS, FMS and
other unexplained painful conditions to be examined for common underlying processes. Altered leptin levels and
a pathological response of the HPA-axis as a result of chronic stress and childhood trauma have been suggested as
one of the driving factors of disease development and severity. Previous studies have demonstrated that methylation
of the leptin promoter can play a regulatory role in addiction. In this study, we hypothesized that methylation of the
leptin promoter is influenced by the degree of childhood traumatization and differs between patients with MSD and
controls. A cohort of 151 patients with MSD and 149 matched healthy volunteers were evaluated using clinical and
psychometric assessment while methylation level analysis of the leptin promoter was performed using DNA isolated
from whole blood.
Results: In female controls, we found CpG C-167 to be negatively correlated with leptin levels, whereas in female
patients CpG C-289, C-255, C-193, C-167 and methylation cluster (C-291 to C-167) at putative bindings sites for tran-
scription factors Sp1 and c/EBPalpha were negatively correlated with leptin levels. Methylation levels were signifi-
cantly lower in female patients CpG C-289 compared with controls. When looking at female patients with chronic
widespread pain methylation levels were significantly lower at CpG C-289, C-255 and methylation cluster (C-291 to
C-167).
Conclusion: Our findings support the hypothesis that epigenetic regulation of leptin plays a role in the regulation of
leptin levels in patients with MSD. This effect is more pronounced in patients with chronic widespread pain.
Keywords: Leptin, Methylation, Multisomatoform disorder, Fibromyalgia, Chronic pain
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Background
In patients presenting with painful symptoms often a
sufficient underlying explanation in terms of a somatic
diagnosis cannot be found. In these cases, the chronic
pain can be characterized as the leading symptom of a
functional somatic syndrome (FSS) such as fibromyalgia
(FMS) or somatoform pain disorder. In such syndromes,
functionally disabling and bothersome physical symp-
toms are also frequently present. is constellation of
symptoms is also present in multisomatoform disorder
(MSD) [1, 2] which is a diagnostic construct to better
characterize these patients across different somatic and
psychological specialties [1, 3]. A diagnosis of MSD can
Open Access
*Correspondence: johannes.achenbach@krh.eu
1 Department of Anesthesiology and Intensive Care Medicine, Nordstadt
Krankenhaus Hannover, Haltenhoffstr. 41, 30167 Hannover, Germany
Full list of author information is available at the end of the article
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Page 2 of 7
Achenbachetal. Clinical Epigenetics (2022) 14:13
be made in the presence of more than three currently dis-
tressing physical symptoms in addition to a long (greater
than 2years) history of somatization. e prevalence of
MSD is 8% and thus posts a relevant disease burden [3]
e pathophysiology of functional somatic syndromes,
fibromyalgia, and MSD is incompletely understood but a
complex interplay of biographic, environmental, genetic,
and epigenetic factors influencing allostasis seems likely
[4, 5], especially as the similarity in symptoms and
patients suggest common mechanisms which lends valid-
ity to the construct of MSD. In a population-based twin
study, genetic influences have been shown to play a role
especially in painful FSS, whereas inconsistent results
suggest a role of single nucleotide polymorphisms (SNPs)
of serotonergic and dopaminergic genes [68]. Our
group recently demonstrated common sensory altera-
tions through quantitative sensory testing in patients
with MSD [9] similar to those found in patients with
fibromyalgia (FMS) [10, 11]. In this context, the construct
of chronic widespread pain is of particular interest. Since
its systematic introduction as part of the diagnostic crite-
ria for FMS in 1990 [12] numerous studies have included
patients with CWP not fulfilling criteria for FMS. How-
ever, different interpretations of the criteria as well as
adaptations over time [13, 14] have made comparisons
not straightforward [15]. DNA methylation describes a
modification through covalent binding of a methyl group
to cytosine residues that are followed by guanine nucle-
otide in the DNA strand (CpG Island). is has among
others been shown to be influenced in a model of early
stress through reduced neonatal maternal care in rodent
models [1618] as well as in chronic pain states [19, 20].
We could also demonstrate the influence of transient
receptor potential ankyrin 1 (TRPA1) receptor promoter
methylation on heat and pressure pain thresholds which
was significantly influenced by the level of childhood
traumatization [21].
Lastly, the complex interplay between obesity in
chronic pain states as well as FMS, leptin and the HPA-
axis has been investigated with growing interest by the
scientific community [22], whereas obesity is a common
comorbidity in FMS and has also been shown to increase
symptom severity [2326], Leptin levels in relation to
painful conditions have been found to be either unal-
tered [27], elevated [2831] or reduced [32] compared
with controls. Leptin is a 16kDa protein predominantly
secreted by adipose tissue or in the brain [33, 34]. Its
main function lies in the regulation of energy homeo-
stasis and conveying a feeling of satiety [3537]. It has
also been shown to have an inhibitory function on the
HPA-axis [38]. In a reverse manner, however, its synthe-
sis is stimulated by cortisol in adipose tissue [39]. Addi-
tionally, leptin has been demonstrated to play a role in
the pathophysiology of neuropathic pain [4042] . e
expression of leptin has been previously shown to be
influenced by epigenetic mechanisms, namely hypometh-
ylation in the promoter region at binding sites for Sp1
and C/EBPalpha [4346] which typically act as activators
of gene expression.
Our group recently demonstrated in the current patient
collective a distinct alteration of the neuroendocrine pro-
file of patients with MSD (publication under review) with
a significantly higher level of leptin and lower levels of
cortisol in female patients compared with controls. We,
therefore, hypothesized that in patients with MSD the
difference in measured leptin levels is influenced by alter-
ations in leptin promoter methylation due to the influ-
ence of childhood trauma.
Materials andmethods
Subjects
Participants in this study have been previously evaluated
with regards to the presence of SNPs of different genes
[68], the presence of sensory alterations using standard-
ized quantitative sensory testing as well as methylation
status of the TRPA1 promoter [9, 21]. Altogether, 151
MSD patients and 149 healthy controls were included in
the study. Patients were recruited through the outpatient
pain clinic of the Hannover Medical School, Hannover,
Germany, and the Clinic for Psychosomatic Medicine
and Psychotherapy of the Hannover Medical School over
a period of 12 months. Additional patients were con-
tacted through local fibromyalgia support groups while
healthy age- and gender-matched participants without
physical pain were included in the control group. Exact
records of the place of recruitment were not kept; most
patients however were partaking in regular treatments
at Hannover Medical School. Severe somatic or psychi-
atric conditions were excluded through expert clinician
assessment while psychometric evaluations through
questionnaires were also performed. All patients’ chief
complaint was chronic widespread pain. Diagnosis of
MSD was supported by means of a modified interview of
the somatoform disorders section of the Structured Clin-
ical Interview for the Diagnostic and Statistical Manual of
Mental Disorder IV (DSM-IV) (SCID) as well as the Ger-
man version of the 36-item Short Form 36 (SF-36) ques-
tionnaire, i.e., the Physical Component Summary score
needed to be 40 as sign of strong psychophysiological
strain [1, 2, 68, 47]. e presence of chronic widespread
pain fulfilling the strict criteria of pain present in three
out of four body quadrants in addition to axial pain [15]
was systematically assessed by a 34-item pain localization
questionnaire.
Exclusion criteria were defined as age < 18years, insuf-
ficient German language ability, insufficient cognitive
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Achenbachetal. Clinical Epigenetics (2022) 14:13
abilities, severe and chronic somatic diseases (e.g.,
severe heart failure, encephalitis disseminata, demen-
tia), and severe comorbid mental disorders which cause
major impairment of social functioning (e.g., schizo-
phrenia, severe mood disorders, personality disorders,
substance abuse) as previously described [68]. Psycho-
metric questionnaires are beyond the scope of the cur-
rent manuscript.
Blood samples were collected and used for DNA
extraction, laboratory, and epigenetic analysis [48, 49].
In all investigations, the revised Declaration of Helsinki
in 2000 (Edinburgh, 52. general meeting) was adhered to
and there was approval by the Ethical Committee of Han-
nover Medical School (study protocol number 4757). All
subjects gave informed consent for blood sampling, gen-
otyping, and clinical measurements [68].
Determination ofLeptin levels
A radioimmunoassay was performed using the human
leptin RIA kit (LINCO Research, St. Charles, Missouri,
USA). Blood was collected between 8.00 and 9.00 am
for each participant to be in keeping with the circadian
rhythm of hormone release of the HPA-axis. EDTA vials
(4ml) and Serum vials (5ml) were used (S-Monovette,
Sarstedt). Measurements were performed through the
Department of Endocrinology of the Hannover Medical
School (MHH).
DNA Isolation
Blood was collected from each subject using two 4-mL
EDTA tubes that were then stored at 80° until extrac-
tion. Genomic DNA from patients and controls was
extracted using a standard high-salt extraction method.
A small subset of DNA samples was isolated by using a
commercially available DNA isolation kit (QiAamp®
blood kit, Qiagen, Hilden, Germany) according to the
manufacturer’s instructions.
Determination ofmethylation rates
DNA was bisulfite-converted using the Epitect conver-
sion kit (Qiagen, Hilden, Germany) according to manu-
facturer recommendations.
Bisulfite-converted DNA was used for PCR amplifi-
cation using specific primer sets (see Additional file1:
Table S1) in a Touchdown PCR approach [50]. Result-
ing amplicons were subjected to linear sequencing PCR
using BigDye Terminator according to manufacturer
instructions (ABI Life Technologies, Grand Island, USA).
For Sequence cleanup prior to sequencing we used
AMPure beads on a Biomek NxP liquid handling plat-
form (Beckman Coulter, Brea, USA). Purified reactions
were sequenced using a 3500xl 24 capillary Sequencer
(ABI Life Technologies, Grand Island, USA).
CpG position is provided in relation to the transcrip-
tional start site located at GRCh38:7:128241278 accord-
ing to ENSEMBL gene accession # ENSG00000174697.
All reported locations are in the proximal promoter
upstream of the gene locus. Sequence analysis and deter-
mination of methylation rates for each CpG site were
conducted using the Epigenetic Sequencing Methyla-
tion analysis software [51]. e methylation rate of each
CpG site per subject was estimated by determining the
ratio between normalized peak values of cytosine and
thymine.
Quality control
Raw sequences were checked for quality and integrity by
using the Sequence Scanner 2 Software (ABI Life Tech-
nologies, Grand Island, USA) and alignment in Geneious
11 (Biomatters, Auckland, New Zealand).
e resulting values were processed further if 95% of
the CpGs of each specimen and 95% of the respective
CpG position were available.
We successfully measured other genes in this collec-
tive (TRPA1) [21] as well as unpublished data. e overall
variance of measured results for TRPA1 and other genes
was very low indicating a high level of precision of the
collected data.
Prediction oftranscription factor binding sites
Potential binding sites for transcription factors (TFs)
were predicted using Geneious 11 (Biomatters, Auckland,
New Zealand) allowing for 1 mismatch base. e find-
ings were confirmed using the Alggen Promo tool (http://
alggen. lsi. upc. es/ cgi- bin/ promo_ v3/ promo/ promo init.
cgi? dirDB= TF_8.3) on the same sequence. Both tools
access the freely available resources at the Transfac pub-
lic database: (http:// gene- regul ation. com/ cgi- bin/ pub/
datab ases/ trans fac/ search. cgi) [52].
Statistical analysis
All statistical calculations were performed using the Sta-
tistical Package for the Social Sciences Version 26 (SPSS,
IBM, Armonk, NY). We used GraphPad Prism for Mac
Version 9 for data illustration (Graphpad Software Inc,
La Jolla, CA). Sequence Scanner v1.0 software (ABI Life
Technologies) was used to assess sequence quality. After
sample quality estimation 151 Patients and 149 controls
were used for data analysis. CpG sites were measured
successfully without need for exclusion from analysis.
Distribution of data was checked according to Shap-
iro–Wilk. For normally distributed data parametric tests
were chosen, in all other instances nonparametric tests
were used. Pearson correlations were used to character-
ize association of methylation with serum leptin levels.
Differences between patients and controls were assessed
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Achenbachetal. Clinical Epigenetics (2022) 14:13
using a two-sided t test for independent samples. Adjust-
ment for multiple comparisons was not made as compar-
isons were preplanned before the study was commenced.
Equality of variance was determined automatically using
the Levene test. Interpretation of the results was con-
ducted accordingly. Results are given as mean and stand-
ard deviation.
Results
Diagnostic criteria for MSD according to the Diagnostic
and Statistical Manual of Mental Disorder-IV (DSM-IV)
were fulfilled by all patients. As previously reported, there
were no differences between gender and age (p > 0.05)
(control group: mean age, 52.1 ± 9.9 years; 73% women
and 27% men; MSD group: mean age, 54.4 ± 10.1 years;
82% women and 18% men) [69, 21]. As expected the
physical component summary score of the SF-36 dem-
onstrated a significant difference between patients and
controls (28.75 ± 7.81 vs 54.0 ± 5.74) (p < 0.0001). e
sample size of male participants proved too small to pro-
vide satisfactory explanatory power. At the same time,
no significant findings could be demonstrated so that
further investigation focused mainly on female study
participants. Leptin measurements were obtained in 244
participants (129 female controls, 91 female patients as
well as 12 male patients and controls each (data submit-
ted for publication).
Methylation
Leptin levels and methylation status at the following
CpGs were negatively correlated: in female controls at
C-167 (rp = 0.205, p = 0.046) and in female patients
at C-289 (rp = 0.232, p = 0.047), C-255 (rp = 0.242,
p = 0.038), C-193 (rp = 0.294, p = 0.022), C-167
(rp = 0.242, p = 0.043) and the mean methylation at
the cluster with binding sites for Sp1, c/EBPalpha and
CREB (C-291 til C-167) (rp = 0.239, p = 0.039). e
observed correlations affected CpGs that were in close
proximity to one another and have been previously
shown to have particular relevance as binding motifs
for Sp1, c/EBPalpha and CREB which are well known to
be involved in the regulation of leptin expression. We,
therefore, decided to further characterize only these
highly thematic CpGs. ere was a significant difference
in the methylation levels of CpG C-289 between female
patients (0.1449 ± 0.9554) and controls (0.1766 ± 0.1000),
t(211) = 2.366, p = 0.019 (see Fig. 1 for most relevant
CpGs). For a graphical representation of the methylation
level at each individual CpG see Additional file2: Fig. S1.
Significant differences between female patients and
controls despite significant correlation with leptin levels
could surprisingly only be found at CpG C-289. To focus
on patients with the highest pain burden, we re-examined
a subset of female patients (120/138) fulfilling the strict
criteria for chronic widespread pain (pain in three out
of four quadrants as well as axial pain). Incomplete data
to determine pain distribution were present in 5 female
controls and 10 female patients. Significant differences
were observed at CpG C-289 (t(182) = 2.990, p = 0.003),
C-255 (t(182) = 2.202, p = 0.029) and methylation cluster
(t(183) = 2.228, p = 0.024). A graphical representation is
given in Fig.2, whereas exact methylation levels are given
in Table1.
Discussion
Hormones regulating dysfunctional responses of the
HPA-axis to chronic stress have been implied in the eti-
ology of most disorders that can be subsumed under the
construct of MSD [5356]. In addition, the role of leptin
and its influence on the HPA-axis and its role with pain-
ful disorders have been investigated. In our study, we
m
ethylation cluster
mean methylation
C-289
C-255
C-193
C-167
0.0
0.1
0.2
0.3
0.4
0.5
Methylation Level
female controls
female patients
Fig. 1 Methylation levels of most relevant CpGs comparing female
patients and female controls. Data represented as mean + 95% CI.
There was a significant difference observed only at CpG C-289
mean methylation
methylation cluste
r
C-289
C-255
C-193
C-167
0.0
0.1
0.2
0.3
0.4
0.5
Methylation Level (mean + 95% CI)
CWP neg.
CWP pos.
Fig. 2 Methylation levels of most relevant CpGs comparing female
patients fulfilling strict criteria for chronic widespread pain and female
controls. Data represented as mean + 95% CI. There was a significant
difference observed CpG C-289, C-255 and methylation cluster
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Achenbachetal. Clinical Epigenetics (2022) 14:13
characterized female patients with MSD in comparison
with healthy controls with regards to the methylation sta-
tus of the leptin promoter region. We focused on female
subjects as women are known to have a higher preva-
lence of MSD [57, 58] because methylation patterns were
found to be gender-dependent in genome-wide associa-
tion studies [59]. We performed a methylation analysis of
the leptin promoter region that revealed significant nega-
tive correlations between methylation at C-289, C-255,
C-193, C-167 and leptin levels in female patients, i.e., less
methylation is correlated with higher leptin levels. is
is plausible as these CpGs are located at binding sites
for transcription factors and higher methylation is often
associated with repressive effects on gene expression
[60]. Transcription factors Sp1 and c/EBPalpha whose
binding is favored in states of reduced methylation and
increases transcription of the gene upon binding to DNA
[6164].
After only observing significantly lower methylation
in CpG-289 in female patients further analysis revealed
that in patients fulfilling strict criteria for CWP had sig-
nificantly lower methylation levels at CpGs -289, -255
and methylation cluster while -167 trended toward sig-
nificance (p = 0.09) It also serves as further support of
our interpretation that lower methylation levels facili-
tate binding of activating transcription factors Sp1 and
c/EBPalpha resulting in higher leptin levels. Previous
studies have demonstrated similar findings in psychiatric
patients suffering from addiction [43]. us, in patients
with MSD methylation at C-289 being significantly lower
can be contributing to observed elevated leptin levels
as this is a known binding site for c/EBPalpha. e lack
of significant differences in CpG -255 and the methyla-
tion cluster could be attributed to lower pain burden in
these patients compared with MSD patients suffering dis-
tinctly from CWP. is is plausible as self-reported pain
has been shown to be associated with leptin levels [31].
Further significant hypomethylation in CpG -255 and
methylation cluster could be a likely corollary, especially
as this is a known binding site of Sp1.
Higher leptin levels in patients with painful condi-
tions are biologically plausible as previous study demon-
strated increased leptin levels in patients with FMS [28,
65] despite other studies showing an opposite effect [32,
66]. Leptin also plays a crucial role in the development
of neuropathic pain in animal models of nerve injury [40,
67] and has been demonstrated to cause allodynia and
hyperalgesia [42] (which are hallmarks of neuropathic
pain conditions but also of central sensitization and noci-
plastic pain). e observation is congruent with the fact
that a subset of patients with FMS shows signs of small
fiber neuropathy [10, 11]. Similar findings have been
previously shown in patients with FMS where BMI and
elevated leptin levels are independently associated with
self-reported pain [31]. Chronic stress is known to cause
a dysregulation of the stress response as mediated by the
HPA-axis [68]; here leptin has been found to play a sig-
nificant role as well [6973] . Taken together our current
findings and the fact that leptin levels are significantly
higher in these female patients with MSD (publication
under review) confer a plausible interrelational connec-
tion with leptin regulation in patients with MSD, espe-
cially with CWP.
One of the limitations of our and other epigenetic stud-
ies is the utilization of DNA from whole blood cells for
analysis. It has been shown that different tissues demon-
strate similar methylation levels [74], other cases have
reported tissue-specific levels [75], whereas neuronal
tissue is preferable, most study designs don’t allow for it
being readily available. A further limitation is the lack of
data on how many possible participants declined to take
part in the study after positive eligibility screening as well
as on location of recruitment (support group, Pain Clinic,
Department of Psychosomatics and Psychotherapy). A
potential for a degree of self-selection bias is however
mitigated by stringent selection criteria that led to a
study population with a high disease burden.
In conclusion, to our knowledge, this is the first study
to thoroughly investigate a large collective of patients
with MSD and pain as the leading symptom with regards
to the epigenetic regulation of leptin expression. Our
study demonstrated that transcriptional regulation is
in part regulated through methylation on an epigenetic
level. Future studies should further validate our results of
site-specific promoter methylation of patients compared
to controls and increased methylation stratified by degree
of widespread pain and stress levels.
Table 1 Mean methylation levels of female patients with
chronic widespread pain (CWP) and female controls without
CWP; SD: standard deviation
CWP negative CWP positive
Mean SD ± Mean SD ±
Mean methylation .2911 .0723 .2800 .0828
Methylation cluster .3270 .0898 .2935 .1106
C-289 .1835 .0987 .1414 .0920
C-255 .3743 .1607 .3205 .1685
C-193 .2522 .1129 .2177 .1481
C-167 .4323 .1849 .3835 .1956
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Achenbachetal. Clinical Epigenetics (2022) 14:13
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13148- 022- 01235-5.
Additional le1: TableS1. Leptin primer list.
Additional le2: Fig. S1. Methylation levels (mean ± 95% CI) of all CpGs
in female patients and female controls.
Acknowledgements
The authors gratefully thank the patients and controls who participated in
this study, and Anh-Thu Tran, Lilly Volkmann, Dennis Buers, Karl Kapitza, Prof.
Michael Bernateck, and Katharina Harms, Jana Jakobi, and Prof. Manfred Stuhr-
mann as well as Nabeela Donaghey for their continuous support.
Authors’ contributions
JA, MR and MK made major contributions to the conception and design of
this work and analyzed and interpreted the data. They were also major con-
tributors in the composition of the manuscript. AG and HF also substantially
contributed to the design of this work and contributed to the writing of the
manuscript. JA, MK and MR were instrumental in the acquisition and analysis
of the data. MR and AG substantially contributed to the interpretation of the
data. All authors have approved the submitted version of the manuscript. All
authors agree to be personally accountable for the manuscript’s content. All
authors read and approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
The study followed the guidelines of the revised UN Declaration of Helsinki
in 2000 (Edinburgh, 52. General Meeting). Following approval by the ethics
committee of the Hannover Medical School (study protocol number 4757),
informed consent was obtained from all patients and controls for blood
sampling, genotyping, and clinical measurements.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Department of Anesthesiology and Intensive Care Medicine, Nordstadt
Krankenhaus Hannover, Haltenhoffstr. 41, 30167 Hannover, Germany. 2 Depart-
ment of Anesthesiology and Intensive Care Medicine, Pain Clinic, Hannover
Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. 3 Laboratory
for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry
and Psychotherapy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Han-
nover, Germany.
Received: 12 April 2021 Accepted: 13 January 2022
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Article
Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder based on a loss of paternally expressed but maternally imprinted genes in chromosome region 15q11-13. PWS individuals typically show insatiable appetite with subsequent obesity representing the major mortality factor unless food intake is inhibited. The neurobiological basis of PWS-typical hyperphagia has remained poorly understood. Many PWS-typical abnormalities are based on hypothalamic dysregulation, a region in which hunger and satiety are hormonally regulated, with the hormone leptin being a main long-term regulator of satiety. Previous studies in PWS have inconsistently shown leptin alterations solely in early childhood, without investigating the leptin system on an epigenetic level. The present study investigates serum leptin levels (S-leptin) and DNA methylation of the leptin (LEP) and leptin receptor gene (LEPR) promoter in 24 individuals with PWS compared to 13 healthy controls matched for sex, age, and body mass index (BMI) and relates the results to the extent of hyperphagia in PWS. S-Leptin levels were obtained by Enzyme-linked Immunosorbent Assay. LEP/LEPR-promoter DNA methylation was assessed by bisulfite-sequencing, hyperphagia by Hyperphagia Questionnaire for Clinical Trials (HQ-CT). PWS and control groups differed significantly in S-leptin levels with higher S-leptin in PWS. Methylation analysis showed significant differences in mean promoter methylation rate both for LEP and LEPR with a lower methylation rate in PWS. LEPR, but not LEP methylation correlated significantly with S-leptin levels. S-leptin and both LEP and LEPR methylation did not correlate with HQ-CT scores in PWS. The present study is the first to show significantly elevated S-leptin levels in an adult PWS cohort combined with an altered, downregulated LEP and LEPR promoter methylation status compared to sex-, age- and BMI-matched controls. Analogous to previous studies, no link to the behavioral dimension could be drawn. Overall, the results suggest an increased leptin dysregulation in PWS, whereby the findings partly mirror those seen in non-syndromic obesity.
Article
Objective Somatoform disorders and functional somatic syndromes (FSS) with symptoms that are not sufficiently explained by physical or technical examination are among the most challenging underlying causes. Many different somatoform disorders and FSS have overlapping symptoms, often with pain as the most prevalent one, leading to a high burden of disease. The concept of multisomatoform disorder (MSD) has been developed to acknowledge that fact. We analyzed a group of 151 patients and 149 matched controls to identify interactions of genetic and environmental factors with a possible influence on the development of MSD. Design In a retrospective case-control study, we performed a statistical analysis on 151 patients and 149 matched controls using logistic regression and a Classification and Regression Tree (CART) analysis. Results The logistic regression analysis of genes and environmental factors demonstrated significant differences in the results of the Trier Inventory of Chronic Stress (TICS) questionnaire, the single nucleotide polymorphism rs1800955 of the dopamine receptor D4 and the single nucleotide polymorphism rs4818 of the enzyme catechol-O-methyltransferase between patients with MSD and healthy controls. The resulting decision tree of the CART analysis determined that the TICS questionnaire was able to differentiate patients and controls most accurately, followed by certain genotypes of the 5-hydroxytryptamine receptor 2A and a single nucleotide polymorphism of the enzyme catechol-O-methyltransferase. Conclusions The results of the statistical analysis identified a gene-environmental interaction possibly leading to MSD. The resulting identifiers could be used as a reference to inform diagnostic algorithms to easier identify patients suffering from MSD.
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Leptin is an adipocytokine that is primarily secreted by white adipose tissue, and it contributes to the pathogenesis of neuropathic pain in collaboration with N-methyl-D-aspartate receptors (NMDARs). Functional NMDARs are a heteromeric complex that primarily comprise two NR1 subunits and two NR2 subunits. NR2A is preferentially located at synaptic sites, and NR2B is enriched at extrasynaptic sites. The roles of synaptic and extrasynaptic NMDARs in the contribution of leptin to neuropathic pain are not clear. The present study examined whether the important role of leptin in neuropathic pain was related to synaptic or extrasynaptic NMDARs. We used a rat model of spared nerve injury (SNI) and demonstrated that the intrathecal administration of the NR2A-selective antagonist NVP-AAM077 and the NR2B-selective antagonist Ro25-6981 prevented and reversed mechanical allodynia following SNI. Administration of exogenous leptin mimicked SNI-induced behavioral allodynia, which was also prevented by NVP-AAM077 and Ro25-6981. Mechanistic studies showed that leptin enhanced NR2B- but not NR2A-mediated currents in spinal lamina II neurons of naïve rats. Leptin also upregulated the expression of NR2B, which was blocked by the NR2B-selective antagonist Ro25-6981, in cultured dorsal root ganglion (DRG) neurons. Leptin enhanced neuronal nitric oxide synthase (nNOS) expression, which was also blocked by Ro25-6981, in cultured DRG cells. However, leptin did not change NR2A expression, and the NR2A-selective antagonist NVP-AAM077 had no effect on leptin-enhanced nNOS expression. Our data suggest an important cellular link between the spinal effects of leptin and the extrasynaptic NMDAR-nNOS-mediated cellular mechanism of neuropathic pain.
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Background: The construct of multisomatoform disorder (MSD) is a common point of reference for patients in different somatic and psychosomatic specialties and therefore useful in studying large well-characterized cohorts of a prototype of a somatoform disorder and in parallel as a functional somatic syndrome (FSS). This disorder is characterized by distressing and functionally disabling somatic symptoms with chronic pain as the most frequent and clinically relevant complaint. Pain is perceived by nociceptive nerve fibers and transferred through the generation of action potentials by different receptor molecules known to determine pain sensitivity in pathophysiological processes. Previous studies have shown that for the transient receptor potential ankyrin 1 (TRPA1), receptor methylation of a particular CpG dinucleotide in the promoter region is inversely associated with both heat pain and pressure pain thresholds. In this study, we hypothesized that TRPA1 promoter methylation regulates pain sensitivity of patients with multisomatoform disorder (MSD). A cohort of 151 patients with MSD and 149 matched healthy volunteers were evaluated using quantitative sensory testing, clinical and psychometric assessment, and methylation analysis using DNA isolated from whole blood. Results: We found CpG -628 to be correlated with mechanical pain threshold and CpG -411 to be correlated with mechanical pain threshold in female volunteers, i.e., higher methylation levels lead to higher pain thresholds. A novel finding is that methylation levels were significantly different between patients with no and severe levels of childhood trauma. CpG methylation also correlated with psychometric assessment of pain and pain levels rated on a visual analog scale. Conclusion: Our findings support the hypothesis that epigenetic regulation of TRPA1 plays a role in mechanical pain sensitivities in healthy volunteers. They further provide evidence for the possible influence of childhood traumatic experiences on the epigenetic regulation of TRPA1 in patients with MSD.
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OBJECTIVE The aim of this study was to investigate whether there is a correlation between serum leptin level, disease activity and inflammation markers in patients with fibromyalgia syndrome (FMS). METHODS A total of 48 patients with FMS diagnosed according to the 1990 American College of Rheumatology criteria were included in the study, as well as 36 healthy women as controls. The Visual Analogue Scale was used to gauge pain severity, the Fibromyalgia Impact Questionnaire was used to assess physical function, the 36-Item Short Form Health Survey was used to examine quality of life, and depression was measured with the Beck Depression Inventory. Blood samples were examined for erythrocyte sedimentation rate (ESR), C-reactive protein level (CRP), high-sensitivity CRP level (hsCRP), the neutrophil-to-lymphocyte ratio (NLR), and the serum leptin level was determined using the enzyme-linked immunosorbent assay method. RESULTS The serum leptin level in patients with FMS was significantly higher than in the healthy group. However, no significant relationship was found between leptin level and clinical and inflammatory parameters. In addition, there were no significant differences between the patients and the control group in measurements of ESR, CRP, hsCRP, or NLR. CONCLUSION A higher serum leptin level in patients with FMS suggested that leptin may play role in the pathogenesis of FMS, yet there was no relationship between leptin and clinical and inflammatory parameters, suggesting that leptin is not an indicator of disease activity in FMS. Additional research should be performed with larger patient groups.
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DNA methylation is the most widely studied of epigenetic mechanisms, with environmental effects recorded through patterned attachments of methyl groups along the DNA that are capable of modifying gene expression without altering the DNA sequencing. The degree to which these patterns of DNA methylation are heritable, the expected range of normality across populations, and the phenotypic relevance of pattern variation remain unclear. Genes regulating metabolic pathways appear to be vulnerable to ongoing nutritional programming over the life course, as dietary nutrients are significant environmental determinants of DNA methylation, supplying both the methyl groups and energy to generate the methylation process. Here we examine methylation patterns along a region of the metabolic gene leptin (LEP). LEP's putative functions include regulation of energy homeostasis, with its signals affecting energy intake and expenditure, adipogenesis and energy storage, lipid and glucose metabolism, bone metabolism, and reproductive endocrine function. A pattern of differential methylation across CpG sites of the LEP core promoter has been previously identified; however, any consistency of pattern or its phenotypic significance is not fully elucidated among populations. Using DNA extracted from unfractionated white blood cells of peripheral blood samples, our pilot study, divided into two parts, examined the significance of variation in DNA methylation patterns along the leptin core promoter in four populations (phase 1) and used biomarkers reflecting leptin's functional process in two of those populations, western Buryat of Siberia and the Mennonite of central Kansas, to investigate the relevance of the ethnic variation identified in the DNA methylation (phase 2). LEP's core promoter region contains both the binding site for C/EBPα (CCAAT/enhancer binding protein alpha), which tempers the final step in adipocyte maturity and capacity to synthesize leptin, and the TATA motif controlling leptin synthesis. Previous studies report that increased methylation in this region is correlated to decreased gene expression, suggesting tissue-specific methylation variation at this region (Melzner et al. 2002). We hypothesized that evidence of nutritional epigenetic programming would be identified through variation in patterns of DNA methylation and that functional relevance of that variation among populations would be identified through biomarkers that reflect leptin's metabolic signals: serum leptin levels, lipoproteins of the lipid transport system, and anthropometric measures.
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Objective Chronic pain is a debilitating condition of multifactorial origin, often without physical findings to explain the presenting symptoms. Of the possible etiologies of persisting painful symptoms, somatoform disorders and functional somatic syndromes (FSS) are among the most challenging, with a prevalence of 8–20%. Many different somatoform disorders and FSS have overlapping symptoms, with pain being the most prevalent one. The concept of multisomatoform disorder (MSD) has been developed to acknowledge that fact. We hypothesized that the concept of MSD will be reflected in a distinct sensory profile of patients compared with healthy controls and possibly provide insight into the type and pathophysiology of the pain commonly experienced by patients. Design We performed comprehensive quantitative sensory testing (QST) in 151 patients and 149 matched controls. Results There were significant differences in the sensory profiles of patients compared with controls. Patients with MSD showed a combination of tactile and thermal hypesthesia combined with mechanical and cold hyperalgesia. This was true for measurements at test and control sites, with the exception of vibration detection threshold and mechanical pain threshold. Among the observed changes, a marked sensory loss of function, as evidenced by an increase in cold detection threshold, and a marked gain of function, as evidenced by a decrease of pressure pain threshold, were most notable. There was no evidence of concurrent medication influencing QST results. Conclusions The observed somatosensory profile of patients with MSD resembles that of patients suffering from neuropathic pain with evidence of central sensitization
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Objective: In healthy individuals, leptin is produced from adipose tissue and is secreted into the circulation to communicate energy balance status to the brain and control fat metabolism. Corticotropin- releasing hormone (CRH) is synthesized in the hypothalamus and regulates stress responses. Among the many adipokines and hormones that control fat metabolism, leptin and CRH both curb appetite and inhibit food intake. Despite numerous reports on leptin and CRH properties and function, little has been actually shown about their association in the adipose tissue environment. Methods: In this article, we summarized the salient information on leptin and CRH in relation to metabolism. We also investigated the direct effect of recombinant CRH on leptin secretion by primary cultures of human adipocytes isolated from subcutaneous abdominal adipose tissue of 7 healthy children and adolescents, and measured CRH and leptin levels in plasma collected from peripheral blood of 24 healthy children and adolescents to assess whether a correlation exists between CRH and leptin levels in the periphery. Results and Conclusion: The available data indicate that CRH exerts a role in the regulation of leptin in human adipocytes. We show that CRH downregulates leptin production by mature adipocytes and that a strong negative correlation exists between CRH and leptin levels in the periphery, and suggest the possible mechanisms of CRH control of leptin. Delineation of CRH control of leptin production by adipocytes may explain unknown pathogenic mechanisms linking stress and metabolism. Keywords: Leptin, corticotropin-releasing hormone (CRH), adipocytes, expression, signaling, stress, metabolism.
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Previous studies have provided evidence of an association between serum leptin levels and smoking as well as craving during smoking cessation. As promoter methylation also regulates leptin expression, we investigated the leptin gene promoter region of smokers before and after smoking cessation. Since leptin's core promoter region contains an essential c/EBPalpha transcription binding site, we narrowed our investigation to C-300 (-300 base pairs from the transcription start site) of that binding site. Female smokers showed hypermethylation of C-300 compared to non-smokers. Global methylation status is associated with higher craving and the degree of dependence in female smokers. Serum leptin levels in female smokers were significantly higher than in non-smokers. These findings support previous results and, for the first time, point to a pathophysiological role of c/EBPalpha-related C-300 methylation in tobacco dependence.
Article
Objective: Hypothalamic-pituitary-adrenal (HPA) axis dysfunction may play a role in fibromyalgia (FM) pathogenesis, but remains understudied in this disorder. Furthermore, early childhood adversities (ECA) are common in FM, but whether they moderate stress reactivity is unknown. Hence, we investigated cortisol and subjective responses to acute psychosocial stress in FM and controls, while adjusting for ECA. Methods: Twenty-seven female FM patients and 24 age-matched female controls were recruited in a tertiary care center and through advertisements, respectively. The Childhood Trauma Questionnaire was used to measure ECA history. Salivary cortisol levels and subjective stress ratings were measured at multiple time points before and after the Trier Social Stress Test (TSST) was administered. Results: Significant main effects of group [F(1,43)=7.04, p=0.011, lower in FM] and ECA [F(1,43)=5.18, p=0.028, higher in participants with ECA] were found for cortisol responses. When excluding controls with ECA (n=5), a significant group-by-time interaction was found [F(6,39)=2.60, p=0.032], driven by a blunted response to the stressor in FM compared with controls (p=0.037). For subjective stress responses, a significant main effect of group [F(1,45)=10.69, p=0.002, higher in FM] and a trend towards a group-by-time interaction effect [F(6,45)=2.05, p=0.078, higher in FM 30 minutes before and 30 and 75 minutes after the TSST, and impaired recovery (difference immediately after - 30 minutes after the TSST) in FM] were found. Conclusions: Blunted cortisol responsivity to the TSST was observed in FM patients compared with controls without ECA. FM patients had higher subjective stress levels compared with controls, particularly at baseline and during recovery from the TSST. In FM patients, ECA history was not associated with cortisol or subjective stress levels, or with responsivity to the TSST. Future research should investigate the mechanisms underlying HPA axis dysregulation in FM.
Article
The brain is the central organ of stress and adaptation to stress that perceives and determines what is threatening, as well as the behavioral and physiological responses to the stressor, and it does so somewhat differently in males and females. The expression of steroid hormone receptors throughout the brain has broadened the definition of 'neuroendocrinology' to include the reciprocal communication between the entire brain and body via hormonal and neural pathways. Mediated in part via systemic hormonal influences, the adult and developing brain possess remarkable structural and functional plasticity in response to stress, including neuronal replacement, dendritic remodeling, and synapse turnover. This article is both an account of an emerging field elucidating brain-body interactions at multiple levels, from molecules to social organization, as well as a personal account of my laboratory's role and, most importantly, the roles of trainees and colleagues, along with my involvement in interdisciplinary groups working on this topic.
Article
Introduction Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it functions as an autocrine hormone. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17β-estradiol up-regulates placental leptin expression through genomic and nongenomic mechanisms. Methods To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, we examined Sp1 transcription factor effect on estradiol leptin induction in human BeWo cell line. Results We demonstrated that Sp1 induces leptin expression determined by qRT-PCR, Western blot and transient transfection experiments. We also found that estradiol induction effect on leptin expression is enhanced by the over expression of Sp1 factor. Moreover, estradiol effect was not evidenced when Sp1 binding site on leptin promoter is mutated, suggesting that estradiol action is dependent on Sp1. On the other hand we showed data that demonstrate that Sp1 induction of leptin expression is insensitive to the antiestrogen ICI 182 780. By over expression experiments, we have also found that Sp1 effect on leptin expression could be mediated by estrogen receptor alpha. Supporting this idea, the downregulation of estrogen receptor alpha level through a specific siRNA, abolished Sp1 effect on leptin expression. Discussion Taken together all these evidences suggest a cooperative behavior between estrogen receptor alpha and Sp1 transcription factors to induce leptin transcription.