Achenbachetal. Clinical Epigenetics (2022) 14:13
Leptin promoter methylation infemale
patients withpainful multisomatoform disorder
andchronic widespread pain
Johannes Achenbach1* , Mathias Rhein3, Alexander Glahn3, Helge Frieling3 and Matthias Karst2
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
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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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
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
Page 2 of 7
Achenbachetal. 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 2years) history of somatization. e prevalence of
MSD is 8% and thus posts a relevant disease burden 
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 [6–8]. Our
group recently demonstrated common sensory altera-
tions through quantitative sensory testing in patients
with MSD  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  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 . 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 [16–18] 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
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 , whereas obesity is a common
comorbidity in FMS and has also been shown to increase
symptom severity [23–26], Leptin levels in relation to
painful conditions have been found to be either unal-
tered , elevated [28–31] or reduced  compared
with controls. Leptin is a 16kDa 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 [35–37]. It has
also been shown to have an inhibitory function on the
HPA-axis . In a reverse manner, however, its synthe-
sis is stimulated by cortisol in adipose tissue . Addi-
tionally, leptin has been demonstrated to play a role in
the pathophysiology of neuropathic pain [40–42] . 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 [43–46] 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.
Participants in this study have been previously evaluated
with regards to the presence of SNPs of diﬀerent genes
[6–8], 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, 6–8, 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 
was systematically assessed by a 34-item pain localization
Exclusion criteria were deﬁned as age < 18years, insuf-
ﬁcient German language ability, insuﬃcient cognitive
Page 3 of 7
Achenbachetal. 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 [6–8]. Psycho-
metric questionnaires are beyond the scope of the cur-
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 [6–8].
Determination ofLeptin 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
(4ml) and Serum vials (5ml) were used (S-Monovette,
Sarstedt). Measurements were performed through the
Department of Endocrinology of the Hannover Medical
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
Determination ofmethylation rates
DNA was bisulﬁte-converted using the Epitect conver-
sion kit (Qiagen, Hilden, Germany) according to manu-
Bisulﬁte-converted DNA was used for PCR ampliﬁ-
cation using speciﬁc primer sets (see Additional ﬁle1:
Table S1) in a Touchdown PCR approach . 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 . e methylation rate of each
CpG site per subject was estimated by determining the
ratio between normalized peak values of cytosine and
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)  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
Prediction oftranscription 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) .
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
Page 4 of 7
Achenbachetal. 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-
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) [6–9, 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).
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 ﬁle2: 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
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 [53–56]. 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
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
Methylation Level (mean + 95% CI)
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
Page 5 of 7
Achenbachetal. 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 . 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
. 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
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 . 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 .
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  (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 . Chronic stress is known to cause
a dysregulation of the stress response as mediated by the
HPA-axis ; here leptin has been found to play a sig-
niﬁcant role as well [69–73] . 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 , other cases have
reported tissue-speciﬁc levels , 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
Page 6 of 7
Achenbachetal. Clinical Epigenetics (2022) 14:13
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13148- 022- 01235-5.
Additional le1: TableS1. Leptin primer list.
Additional le2: Fig. S1. Methylation levels (mean ± 95% CI) of all CpGs
in female patients and female controls.
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.
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.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
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
The authors declare that they have no competing interests.
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-
Received: 12 April 2021 Accepted: 13 January 2022
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