Genetic vulnerability to DUSP22 promoter hypermethylation is involved in the relation between in utero famine exposure and schizophrenia

Abstract

Epigenetic changes may account for the doubled risk to develop schizophrenia in individuals exposed to famine in utero. We therefore investigated DNA methylation in a unique sample of patients and healthy individuals conceived during the great famine in China. Subsequently, we examined two case-control samples without famine exposure in whole blood and brain tissue. To shed light on the causality of the relation between famine exposure and DNA methylation, we exposed human fibroblasts to nutritional deprivation. In the famine-exposed schizophrenia patients, we found significant hypermethylation of the dual specificity phosphatase 22 (DUSP22) gene promoter (Chr6:291687-293285) (N = 153, p = 0.01). In this sample, DUSP22 methylation was also significantly higher in patients independent of famine exposure (p = 0.025), suggesting that hypermethylation of DUSP22 is also more generally involved in schizophrenia risk. Similarly, DUSP22 methylation was also higher in two separate case-control samples not exposed to famine using DNA from whole blood (N = 64, p = 0.03) and postmortem brains (N = 214, p = 0.007). DUSP22 methylation showed strong genetic regulation across chromosomes by a region on chromosome 16 which was consistent with new 3D genome interaction data. The presence of a direct link between famine and DUSP22 transcription was supported by data from cultured human fibroblasts that showed increased methylation (p = 0.048) and expression (p = 0.019) in response to nutritional deprivation (N = 10). These results highlight an epigenetic locus that is genetically regulated across chromosomes and that is involved in the response to early-life exposure to famine and that is relevant for a major psychiatric disorder.

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ARTICLE OPEN
Genetic vulnerability to DUSP22 promoter hypermethylation
is involved in the relation between in utero famine exposure
and schizophrenia
M. P. Boks
1
, L. C. Houtepen
1
,Z.Xu
1
,Y.He
1
, G. Ursini
2
, A. X. Maihofer
3,4,5
, P. Rajarajan
6
,Q.Yu
7
,H.Xu
7
,Y.Wu
7
, S. Wang
7
,J.P.Shi
7
,
H. E. Hulshoff Pol
1
, E. Strengman
8
, B. P. F. Rutten
9
, A. E. Jaffe
2
, J. E. Kleinman
2
, D. G. Baker
3,4,5
,E.M.Hol
10
, S. Akbarian
6
,
C. M. Nievergelt
3,4,5
, L. D. De Witte
1
, C. H. Vinkers
1
, D. R. Weinberger
2
,J.Yu
7
and R. S. Kahn
1,6
Epigenetic changes may account for the doubled risk to develop schizophrenia in individuals exposed to famine in utero. We
therefore investigated DNA methylation in a unique sample of patients and healthy individuals conceived during the great famine
in China. Subsequently, we examined two case-control samples without famine exposure in whole blood and brain tissue. To shed
light on the causality of the relation between famine exposure and DNA methylation, we exposed human fibroblasts to nutritional
deprivation. In the famine-exposed schizophrenia patients, we found significant hypermethylation of the dual specificity
phosphatase 22 (DUSP22) gene promoter (Chr6:291687-293285) (N=153, p=0.01). In this sample, DUSP22 methylation was also
significantly higher in patients independent of famine exposure (p=0.025), suggesting that hypermethylation of DUSP22 is also
more generally involved in schizophrenia risk. Similarly, DUSP22 methylation was also higher in two separate case-control samples
not exposed to famine using DNA from whole blood (N=64, p=0.03) and postmortem brains (N=214, p=0.007). DUSP22
methylation showed strong genetic regulation across chromosomes by a region on chromosome 16 which was consistent with new
3D genome interaction data. The presence of a direct link between famine and DUSP22 transcription was supported by data from
cultured human fibroblasts that showed increased methylation (p=0.048) and expression (p=0.019) in response to nutritional
deprivation (N=10). These results highlight an epigenetic locus that is genetically regulated across chromosomes and that is
involved in the response to early-life exposure to famine and that is relevant for a major psychiatric disorder.
npj Schizophrenia (2018) 4:16 ; doi:10.1038/s41537-018-0058-4
INTRODUCTION
Schizophrenia is a severe psychiatric disorder with a global life-
time risk of around 1% and a typical onset in late adolescence and
early adulthood. In addition to a pronounced polygenic compo-
nent,
1
several environmental risk factors have been identified,
2
of
which prenatal famine is one of the strongest: an almost two-fold
increase was reported in offspring conceived during the Dutch
hunger winter in 1945
3
and at the time of the Great Chinese
famine (1959–1961).
4–6
The mechanism underlying the relationship between famine
exposure and schizophrenia risk remains unclear, but emerging
evidence suggests that epigenetic reprogramming in response to
famine exposure may play a role. Indeed, famine exposure in the
first trimester of pregnancy leads to DNA methylation changes
and these in turn have been found to be related to cardiovascular
disorders.
7–11
However, the relationship between famine-induced
epigenetic changes and schizophrenia has not been studied.
We hypothesized that changes in DNA methylation play a role
in the increased risk to develop schizophrenia after in utero
exposure to famine. To test this hypothesis, we focused on the
great famine in China between 1959 and 1961, which led to an
estimated death toll of over 30 million.
12
The high penetrance of
famine in a large rural population during a restricted period offers
an opportunity for selective sampling of schizophrenia patients
and healthy controls on the basis of their exposure to famine. We
also studied the role of the identified DNA methylation marks in
blood and brain DNA samples of unexposed schizophrenia
patients and controls. Moreover, we carried out in vitro experi-
mental studies whereby human fibroblasts were exposed to
nutritional deprivation to directly investigate methylation
responses to nutritional deprivation without the potential
confounds of genetic differences, medication, and other environ-
mental factors.
Contemporary studies of DNA methylation show that much of
the variability in DNA methylation is controlled by genetic
Received: 09 January 2018 Revised: 29 June 2018 Accepted: 03 July 2018
1
Brain Center Rudolf Magnus, Department of Psychiatry, University Medical Center Utrecht, Utrecht, The Netherlands;
2
Lieber Institute for Brain Development, Johns Hopkins
Medical Campus, Baltimore, USA;
3
Department of Psychiatry, University of California, La Jolla, San Diego, CA, USA;
4
VA Center of Excellence for Stress and Mental Health, San
Diego, CA, USA;
5
Veterans Affairs San Diego Healthcare System, San Diego, CA, USA;
6
Departments of Psychiatry and Neuroscience, Icahn School of Medicine at Mount Sinai, New
York, USA;
7
Department of Epidemiology and Health Statistics, School of Public Health, Jilin University, Changchun, China;
8
Molecular Pathology, Department of Pathology,
University Medical Center Utrecht, Utrecht, The Netherlands;
9
School for Mental Health and Neuroscience, Department of Psychiatry and Neuropsychology, Maastricht University
Medical Centre, Maastricht, The Netherlands and
10
Brain Center Rudolf Magnus, Department of Translational Neuroscience, University Medical Center Utrecht, Utrecht, The
Netherlands
Correspondence: M P. Boks (m.p.m.boks@umcutrecht.nl)
www.nature.com/npjschz
Published in partnership with the Schizophrenia International Research Society

variation in the same genetic region (in cis)
13
as well as in genetic
regions more distant from the methylation mark (in trans).
14
Another point of increasing interest is the relation of the DNA
methylation differences with gene expression, as a relevant
functional readout of methylation differences.
15
Further analyses
therefore investigated the relationship of identified methylation
differences with genotypes as well as their relations with
expression.
RESULTS
Four samples were included in the current paper. (1) The Chinese
famine sample in which the relation between famine and
schizophrenia was studied. (2) A case-control blood sample from
the Netherlands in which we analyzed differences between
unexposed schizophrenia patients and unaffected individuals
using DNA from whole blood. (3) The case-control brain sample in
which we replicated case-control differences using DNA from
brain tissue and analyzed the relations with genotype and gene
expression. (4) Fibroblasts cultures obtained from schizophrenia
patients and healthy controls. Table 1gives an overview of the
characteristics of these four samples.
The genome-wide methylation analysis of the Chinese famine
sample identified one single region containing the dual specificity
phosphatase 22 (DUSP22) gene promoter with higher DNA
methylation levels in the famine-exposed schizophrenia patients
compared to all other groups (Chr6: 291687-293285, Family Wise
Error Rate (FWER) =0.01). DUSP22 methylation was also signifi-
cantly higher in Chinese schizophrenia patients independent of
famine exposure (B=0.07, p=0.025). DUSP22 hypermethylation
in the same region was also significant in blood DNA samples of
Dutch schizophrenia patients (N=15, mean methylation =0.43,
sd =0.10) compared to healthy controls (N=49, mean methyla-
tion =0.32, sd =0.20, p=0.03). DUSP22 methylation was also
higher in postmortem prefrontal cortex (PFC) tissue of schizo-
phrenia patients (N=91; mean methylation =0.40, sd =0.10)
compared to unaffected controls (N=123; mean methylation =
0.37, sd =0.13) (B=0.35, p=0.007). Figure 1and Table 2show
the results from the association analysis.
Genetic control of DUSP22 DNA methylation
In the brain case-control sample, the association between genetic
loci and methylation levels at the ten loci (CpGs) in the DUSP22
differentially methylated region (DMR) was examined for 7.5
million observed +imputed single nucleotide polymorphisms
(SNPs) with minor allele frequency (MAF >5%). We identified 69
SNPs, all on chromosome 16, that were associated with all ten
methylation loci at the p<10
–20
significance level (see supple-
mentary table 1). This genetic regulation outside the DUSP22
region (in trans) is consistent with previous studies
14
and three
online databases of genetic variants associated with methylation
methylation Quantitative Trait Locus (mQTLs) for either fetal brain
tissue (http://epigenetics.essex.ac.uk/mQTL/
16
) or whole blood
(http://genenetwork.nl/biosqtlbrowser/
17
and http://www.mqtldb.
org/
18
), reporting five trans-SNPs on chromosome 16 (rs1433753,
rs9674439, rs12923277, rs12927233, and rs12933929). The under-
lying genetic background spans a region on chromosome 16
(Chr16:34190042-46441560) as large as 12 Mb, and the previously
reported five trans-SNPs are at least 15,000 base pairs away from
each other. It is worth noticing that data on chromosome
interactions (obtained using Hybridization Capture—Hi-C) in
postmortem neurons confirm the strong interaction between
the DUSP22 DMR and the mQTL region on chromosome 6. The
strongest interaction is present between the 3′end on chromo-
some 16 (chr16:34160000-34200000) and the region chr6:280000-
320000 (see Fig. 2a, b). Visual inspection of the data of Rao et al.
19
from human GM12878 B-lymphoblastoid cells also indicates that
the entire region containing these trans-SNPs is in interchromo-
somal contact with the DUSP22 promoter region (see supple-
mental data 1). Subsequent mixture analysis identified three
underlying Gaussian distributions in the samples of the study (see
Fig. 3) that we used as an indicator of genetic determinant of the
methylation levels, subsequently coined “genetic background”.
Influence of genotype on schizophrenia risk
The Chinese famine sample showed a significant
gene–environment interaction between the genetic background
and famine exposure on schizophrenia risk (B=1.20, p=0.042).
After adjustment for genetic background, the relationship of
DUSP22 methylation with famine and schizophrenia was only
present in a selection of participants without the 35 participants
with a genetic predisposition for low invariable methylation levels
(N=118, B=0.028, p=0.072). In the Dutch case-control sample,
the association between DUSP22 promoter methylation and
schizophrenia persisted after adjustment for genetic background
(B=0.741, p=0.025). In the postmortem brains, the association
between DUSP22 promoter methylation and schizophrenia was
attenuated after adjustment for genetic background (B=0.13, t=
1.745, p=0.083).
Ethnicity
Ethnicity influenced the relationship between DUSP22 methylation
and schizophrenia in the brain case-control sample (Schizophrenia
by Race interaction: B=0.680, p=0.007). The genetic background
of DUSP22 methylation was significantly different between
African–American and Caucasian subjects (p< 0.001 in Pearson’s
χ
2
test). The absence of the low methylation genotype in the
African–Americans contributes to the lower variation in DUSP22
Table 1. Sample characteristics of study samples
Sample 1: Chinese famine Sample 2: Case-control
blood
Sample 3: Case-control brain Sample 4: Fibroblasts
Schizophrenia Controls Schizophrenia Controls Schizophrenia Controls Schizophrenia Controls
N74 79 15 49 91 123 5 5
Male (%) 46 (62%) 31 (39%) 9 (60%) 4 (14%) 55% 67% 3 (60%) 2 (40%)
Mean age (sd) 47.3 (0.7) 47.9 (0.8) 40.1 (13.8) 35.9 (17.0) 52.6 (5.2) 45.9 (16.8) 39.0 (10.3) 36.5 (6.5)
Famine exposure (%) 23 (31%) 25 (32%) ––––In vitro In vitro
Tissue source Blood Blood Brain (DLPFC) Fibroblast culture
Methylation 450K BeadChip array 450K BeadChip array 450K BeadChip array EPIC BeadChip array
Reference workflow –– Jaffe et al.
4
–
SCZ schizophrenia, DLPFC dorsolateral prefrontal cortex
Genetic vulnerability to DUSP22 promoter hypermethylation is. . .
MP Boks et al.
2
npj Schizophrenia (2018) 16 Published in partnership with the Schizophrenia International Research Society
1234567890():,;

Fig. 1 Identification and replication of a differentially methylated region in DUSP22 in blood. Overview of the 3000 bp area downstream and
upstream of the dual specificity phosphatase 22 (DUSP22) differentially methylated region (DMR). The top panel displays the blood DNA
methylation levels per group in the Chinese famine discovery sample (first panel). The second and third panels contain the DNA methylation
levels for schizophrenia patients and healthy controls in blood or brain tissue, respectively. The other panels indicate the presence of coding
exons (blue blocks) and non-coding introns (gray line) of the DUSP22 gene (fourth panel), and the location of a CpG island (fifth panel) based
on information extracted for genome build Hg19 from the UCSC website41 with the gviz R package 42. The DUSP22 DMR is indicated across
all panels with a light-blue rectangle; chr, chromosome
Table 2. Detailed data by exposure and schizophrenia status
Chinese famine cohort
Schizophrenia Controls
Exposed Unexposed Exposed Unexposed
N23 51 25 54
Age 50.1 (0.6) 46.7 (0.8) 50.3 (0.5) 46.8 (1.0)
Male (%) 18 (69%) 28 (54%) 10 (40%) 21 (39%)
DUSP22 Methylation (sd) 0.46 (0.04) 0.35 (0.17) 0.31 (0.19) 0.33 (0.18)
Case-control blood cohort
Schizophrenia Controls
N15 49
Age, mean (sd) 40.1 (13.8) 35.9 (17.0)
Male (%) 60% 10%
DUSP22 Methylation (sd) 0.43 (0.10) 0.32 (0.20)
Case-control brain cohort
Schizophrenia Controls
N91 123
Age, mean (sd) 52.6 (5.2) 45.9 (16.8)
Male (%) 55% 67%
DUSP22 Methylation (sd) 0.40 (0.10) 0.37 (0.13)
Genetic vulnerability to DUSP22 promoter hypermethylation is. . .
MP Boks et al.
3
Published in partnership with the Schizophrenia International Research Society npj Schizophrenia (2018) 16

DMR methylation levels (see Table 3). This is in line with the
stratified analyses for ethnicity that show an association between
DUSP22 methylation and schizophrenia in the Caucasians only (B
=0.692, p=0.002), and not in the African–Americans (B=−0.086,
p=0.436).
Smoking and urban background
In the Chinese famine sample, as expected, smoking was more
frequent in the schizophrenia patients (B=0.01, p=0.046). No
differences between the famine and non-famine groups were
present (p=0.9). The DUSP22 promoter methylation was also not
associated with the smoking proxy (B=0.028, p=0.112), nor is it
highlighted in previous association studies into smoking.
20,21
Also
inclusion of urban background in the model did not alter the
results. In the case-control blood samples, five of 15 schizophrenia
patients were current smokers in contrast to three of 49 smokers
in the healthy controls (χ
2
test, p=0.02), DUSP22 promoter
methylation was not associated with schizophrenia status (B=
−0.79, p=0.223). Smoking was dealt with in the case-control
brain samples by adjusting the methylation data using the first
principle components.
22
Supplemental data Figs. 1,2, and 4 show
the relations of smoking with the variables of interest.
DUSP22 methylation and expression
A correlation between DUSP22 methylation and expression was
not present in the brain samples, nor was genetic background
Fig. 2 Overview of the chromosome–chromosome interactions measured with in situ Hi-C. Panel azooms into the DUSP22 DMR, while b
provides an overview of the chromosome interactions. A darker blue indicates more frequent interactions
Fig. 3 Density plot of the average methylation at the DUSP22 differentially methylated region (DMR) in the four population samples: the
Chinese famine sample, the case-control blood sample, the blood genomics sample, and the brain case-control sample. The colored lines
represent the estimation of the underlying distributions in the respective samples
Genetic vulnerability to DUSP22 promoter hypermethylation is. . .
MP Boks et al.
4
npj Schizophrenia (2018) 16 Published in partnership with the Schizophrenia International Research Society

associated with DUSP22 expression. In contrast, the schizophrenia
patients had significantly lower DUSP22 transcript levels (schizo-
phrenia: N=60, DUSP22 expression =2.97, sd =0.17; controls: N
=96, DUSP22 expression =2.87, sd =0.21) (B=−0.09, p=0.005),
also after adjustment for genetic background (B=−0.14, p<
0.001), suggesting transcriptional regulation of DUSP22 by other
factors than DNA methylation in the adult brain of schizophrenia
patients.
Fibroblast
Depriving fibroblasts from nutrition by withholding 15% fetal
bovine serum (FBS) resulted in a significant increase in DUSP22
methylation (Paired Wilcoxon, p-value =0.049) and an almost
two-fold increase in DUSP22 expression after 72 h (Paired
Wilcoxon, p-value =0.019). Analysis of the methylation difference
between the famine and control conditions showed no significant
difference in response comparing fibroblasts from schizophrenia
patients and controls. Removal of one member of the included
healthy homozygous twin pair slightly reduced the significance (p
=0.054 or p=0.063 depending on which member was removed).
DISCUSSION
Genome-wide analysis of DNA methylation in whole blood from a
sample of schizophrenia patients (N=74) and controls (N=79),
where one-third of both groups were exposed in utero to the
great famine in China (1959–1961), identified one region in the
promoter of the dual specificity phosphatase 22 (DUSP22) gene
(Chr6:291687-293285) with significantly higher DNA methylation
levels in famine-exposed schizophrenia patients compared to
unexposed patients and healthy controls. In this sample, patients
also had significant hypermethylation independent of famine
exposure suggesting that DUSP22 hypermethylation is primarily
involved in schizophrenia. In an independent but unexposed
Dutch sample, a similarly unbiased genome-wide analysis of
whole-blood DNA identified the same hypermethylated DUSP22
region in schizophrenia patients (N=15) as compared to healthy
individuals (N=49). In the postmortem tissue from the PFC,
significant DUSP22 hypermethylation was also found in schizo-
phrenia patients (N=91) compared to unaffected individuals (N
=123). Support for a direct relationship between famine exposure
and DUSP22 methylation was obtained by depriving the
fibroblasts of schizophrenia patients (N=5) and controls (N=5)
from nutrition. DUSP22 methylation and expression significantly
increased in response to nutritional deprivation.
The hypermethylated region in DUSP22 encompasses a CpG
island in the promoter as well as the histone marks H3K27ac and
H3K4me3 that are indicative of active gene transcription in both
PFC brain and blood cells.
23,24
This region contains ten CpG loci
and showed clustering (banding) of the methylation levels
indicative of genetic regulation. In the postmortem brains, we
identified 69 SNPs on chromosome 16 with highly significant
associations with DUSP22 methylation, which included previously
reported mQTLs in blood
17
and brain.
16
This interaction between
the DUSP22 DMR on chromosome 6 and the SNPs on chromosome
16 (Chr16:34190042-46441560) is consistent with new data of Hi-C
proximity maps from human postmortem brains as well as
previously published Hi-C data on human lymphoblastoid cells.
19
These data highlight the importance of SNPs that physically
interact in three-dimensions (3D) with chromatin and influence
target transcript levels.
14
The identified mQTLs are not associated
with schizophrenia in the most recent GWAS meta-analysis
4
suggesting that these genetic variants are not primary risk alleles
for schizophrenia.
The biological relevance of DUSP22 methylation could not be
substantiated by a correlation between DUSP22 methylation and
expression in the brain samples, nor was genetic background
associated with DUSP22 expression. In the brain samples, the
schizophrenia patients had significantly lower DUSP22 transcript
levels uncorrelated with DUSP22 methylation and genetic back-
ground suggesting that in the adult brain of schizophrenia
patients, transcriptional regulation of DUSP22 is independent of
DNA methylation or that other factors interfere with the relation-
ship between DUSP22 methylation and expression. There are
several factors that may confound the reported relationships, and
although we provided replication and have investigated several
potential confounders including smoking, the relatively small
sample sizes and residual confounding remain a limitation. Also
urban origin was defined by the current dwelling, and it is possible
that it is not an accurate reflection of the birthplace.
The results of this series of experiments suggest that altered
transcriptional regulation of DUSP22 in response to famine is a
schizophrenia susceptibility factor. DUSP22 is a recently identified
Dual Specificity Phosphatase. Studies on the hippocampus of
patients with Alzheimer’s disease have linked promoter hyper-
methylation of DUSP22 to changes in TAU phosphorylation,
25
which in turn has been connected to nutritional deprivation.
26
A
further link between DUSP22 methylation and nutrition is
supported by trial data showing that low DUSP22 DNA methyla-
tion at baseline predicted high weight loss in response to a dietary
intervention.
27
Notwithstanding these pre-existing links between DUSP22
methylation and nutrition, the presence of higher DUSP22
methylation in the blood and brain of schizophrenia patients
not exposed to famine also suggest that aberrant DUSP22
methylation is more generally involved in the neurodevelop-
mental processes underlying the etiology of schizophrenia. The
absence of any signal of DUSP22 methylation in recent large EWAS
studies in schizophrenia
28–32
and the absence of an association
with the genetic variation associated with these methylation
differences
4
points out that DUSP22 is not a primary risk gene for
schizophrenia. Instead the evidence from this and other studies
point to a role of DUSP22 methylation in regulating responses to a
variety of environmental stressors
33–36
that may deviate early
developmental processes of the brain.
37,52
It is possible that such
vulnerability in combination with particular environmental insults
increase the risk of schizophrenia in a subgroup. In support, a
recent study by Vitale et al.
38
showed that one of the CpG in our
DMR (cg11235426, Chr6: 292522) was differentially methylated in
induced pluripotent stem cells (iPSCs) from schizophrenia patients
as compared to controls (logFC =−2.44, FDR =0.04) and in
schizophrenia patients prenatally exposed to diethylstilbestrol
Table 3. Sample characteristics of the brain dataset per ethnicity
African–American Caucasian All
N99 115 214
Age (mean (sd)) 49.24 (16.30) 48.28 (16.62) 48.73
(16.44)
Male (%) 58 (58.6) 74 (64.3) 132 (61.7)
Schizophrenia (%) 38 (38.4) 53 (46.1) 91 (42.5)
DUSP22 DMR (mean (sd)) 0.43 (0.07) 0.34 (0.14)* 0.38 (0.12)
DUSP22 expression
(mean (sd))
2.83 (0.33) 2.81 (0.33) 2.82 (0.33)
Genetic background (%)
1 3 (3.1) 27 (23.5)* 30 (14.0)
2 35 (36.4) 65 (56.5)* 100 (46.7)
3 61 (63.5) 23 (20.0)* 84 (39.3)
*p< 0.001 in either a Pearson’sχ
2
test (for genotype) or t-test (for
methylation levels)
Genetic vulnerability to DUSP22 promoter hypermethylation is. . .
MP Boks et al.
5
Published in partnership with the Schizophrenia International Research Society npj Schizophrenia (2018) 16

(DES), DUSP22 methylation (Chr6:291687-293331) was higher
compared to exposed controls (unadjusted p-value =0.00018).
39
Further, of note is the strong trans genetic regulation that
stretches over 30 Mb on a different chromosome that coincides
with chromosome-chromosome interactions.
19
The data fita
model where genetic background determines environmental
susceptibility of the DUSP22 gene and schizophrenia risk. Famine
changes the epigenetic regulation of the DUSP22 promoter in
those that are genetically vulnerable. This putative epigenetic
mechanism of early-life environmental influences on brain
development is likely to be important, not only for understanding
the etiology of schizophrenia, but also because it opens new
perspectives on mechanisms of gene–environment interactions.
MATERIAL AND METHODS
Chinese famine sample
The Chinese famine started suddenly in 1959 as the result of
agriculture reforms by Mao and lasted until 1961. Whereas the
onset was sudden, the exact end dates vary by geographical
location due to a subsequent drought that affected the northern
provinces.
40
In collaboration with the University in Changchun in
the Northern Province of Jilin, we included schizophrenia patients
and healthy controls that had been exposed to famine within the
first 3 months of gestation based on a birth date between January
1960 and September 1961. All participants gave written informed
consent. In order to balance the potential influence of urban
background,
5
recruitment was stratified for rural and city hospitals.
A total of 74 schizophrenia patients and 81 healthy controls
approximately matched for famine exposure were assessed.
Information on medication was obtained using a structured
interview. Diagnosis was made using a full psychiatric evaluation
according to DSM IV criteria by licensed psychiatrists. Patients
diagnosed with schizophrenia according to DSM IV included those
with 295.xSchizophrenia, 295.4 Schizophreniform disorder, 295.7
Schizoaffective disorder, 297.1 Delusional disorder, but excluded
298.8 Brief psychotic disorder, 297.3 Shared psychotic disorder,
293.xPsychotic disorder due to a medical condition, 293.x
Substance-induced psychotic disorders, and 298.9 Psychotic
disorder not otherwise specified. The absence of a mental health
disorder in the healthy controls was assessed by a Chinese
translation of the Mental Health Screening Form-III (MHSF-III).
41,42
If more than 10% of the patients used a specific medication type,
we investigated its association with DNA methylation (using the
first principal component of the methylation measures). Based on
this criterion, clozapine (n=37) and chlorpromazine (n=26) use
were examined as potential confounders in the Chinese famine
discovery sample by analyzing the correlation with main
determinants (famine and diagnosis) and outcome (methylation).
Smoking was addressed similarly as to Hannon et al.
43
whereby a
smoking proxy was calculated based on DNA methylation values
for CpGs previously associated with smoking.
20,21
In cases where
these correlations were significant and changes in the coefficient
were larger than 10%, the variable was considered a potential
confounder and models were adjusted by including it as covariate
(see Extended Data Fig. 1). Based on their correlation with general
DNA methylation levels (Extended Data Fig. 2), covariates
included: age, gender, the first two DNA methylation-based
ancestry principal components as well as the cell-type proportion
estimates based on the Houseman algorithm.
44
We separately
investigated the potential role of urban versus rural background
by including this as covariate in the analysis. We excluded one
sample based on gender mismatch with the gender prediction
from DNA methylation levels (one non-famine-exposed healthy
control) and one sample based on the use of insulin (one famine-
exposed healthy control).
Case-control blood samples
Whole-blood DNA samples were collected in 2016 from 15 schizo-
phrenia patients (9 male, mean age =40.1, sd =13.8) and 49
healthy controls (4 male, mean age =35.9, sd =17.0) at the
University Medical Center, Utrecht, The Netherlands. Participants
were of Dutch origin with three or more Dutch grandparents.
Patients were outpatients from the University Medical Center.
Eligibility was assessed by their treating psychiatrist and inclusion
was done by research staff (LH). Healthy controls were recruited in
the general population using online and paper advertisements. All
participants gave written informed consent. Diagnosis of schizo-
phrenia (295.x)(N=11) or schizoaffective disorder (295.7) (N=4)
according to DSM IV criteria was verified in medical records,
supplied by the treating physician or established with the
Structured Clinical Interview for DSM-IV (SCID).
45
In the healthy
controls, the absence of a DSM-IV diagnosis was assessed with the
Mini International Neuropsychiatric Interview (MINI) plus
46
by at
least one well-trained rater. Medication use was collected using a
self-report questionnaire. All schizophrenia patients were on a
stable (at least 1 month) dosing schedule of psychotropic
medication.
Case-control brain samples
We investigated DUSP22 DNA methylation, mRNA, and genotype
in 214 adult postmortem dorsolateral PFC samples of patients with
schizophrenia according to DSM-IV (N=91, 50 male, mean age =
52.6, sd =5.2) and unaffected controls (N=123, 82 male, mean
age =45.9, sd =16.8) from the Lieber Institute.
22
All participants
gave written informed consent. A majority was Caucasian
(Patients: N=53, 27 male, age =50.4 ± 15.6; Controls: N=62, 47
male, age =46.4 ± 17.4) but a sizable proportion (46%) was of
Afro-American descent (Patients: N=38, 23 male, age =55.5 ±
14.2; Controls: N=61, 35 male, age =45.3 ± 16.4). Postmortem
diagnosis according to DSM-IV was obtained when two board-
certified psychiatrists reached consensus after reviewing data
from as many sources as possible (i.e., multiple psychiatric records,
police reports, neuropathology reports, medical examiner’s
information, toxicology screen, postmortem family interview).
47
Medication was assessed via a chart review and/or toxicology on
brain tissue.
22
Fibroblast cell lines
Fibroblast cell lines were established using skin biopsies from five
schizophrenia patients (2 male, mean age =39.0, sd =10.3) and
five age-matched healthy controls (1 male, mean age =38.4, sd =
7.0). All participants gave written informed consent. One healthy
control donor (control 6) turned out to be the homozygous co-
twin of another (control 8). All participants were Dutch and had
three or more Dutch grandparents. Schizophrenia (295.x) was
diagnosed according to DSM-IV as established using the
Comprehensive Assessment of Psychiatric symptoms and History
(CASH).
48
The mental health status of healthy controls was
checked using the MINIplus interview.
46
General analysis
All data were obtained after written, informed consent from all
participants and local medical ethical approval. This research was
conducted in accordance with all relevant guidelines and
procedures, and the work was approved by the University Medical
Center medical ethical review board. Statistical analyses were
carried out using R version 3.2.3.
49
For DNA methylation, βvalues
(the ratio between methylated and unmethylated probe inten-
sities as a measure of methylation percentage) were used for
graphical display and reporting because they are more intuitive,
but analyses were carried out using M-values (log2 of βvalues),
which have better statistical validity,
50
but give similar results.
Genetic vulnerability to DUSP22 promoter hypermethylation is. . .
MP Boks et al.
6
npj Schizophrenia (2018) 16 Published in partnership with the Schizophrenia International Research Society

Genome wide analysis of DNA methylation
All details of quality control, batch effect removal and analysis are
reported in the supplemental materials. In the Chinese famine and
the case-control blood sample, quality control included filtering
for detection of p-values, low bead count, cross hybridizing- and
non-autosomal probes. Normalization was done using functional
normalization as implemented in the minfiR package.
51
Batch
effects were limited as a result of the distribution of the samples of
the array; we only removed a small remaining batch effect for
position. Methylation levels were adjusted for cell-type composi-
tion estimates derived using the Houseman algorithm.
44
Linear
regression was used to identify associations of single CpG with
famine and schizophrenia and the bumphunter algorithm
52
for
differential methylated regions (details in supplement).
The quality control and analysis of the brain case-control
sample were described previously
22
and include adjustment for
neuronal proportion and adjustment for technical batches.
Banding and trans genetic regulation of DUSP22 methylation
At the DUSP22 DMR, there were different DNA methylation bands
in all four population samples: the Chinese famine sample, the
case-control blood sample, the blood genomics sample, and the
brain case-control sample (see Fig. 1). Measurement errors due to
genetic variants in cis that can interfere with hybridization
13,53
were ruled out by showing that five out of 12 SNPs that are
located on the DNA sequence of the DUSP22 DMR in the
dbSNP142 database and that were available in both the MRS and
brain case-control samples (rs860102, rs3734780, rs117766562,
rs9503164, and rs148619589) had a minor allele frequency (MAF)
<5% and were not associated at the 5% level with DUSP22 DMR
methylation in either the blood genomics samples or the brain
case-control sample. Therefore, we did not examine these cis-SNPs
any further. To examine the influence of genetic background on
DNA methylation levels, we investigated the association of the
mean methylation of the ten CpG in the DUSP22 DMR with
genotype. Moreover, we used finite mixture modeling as
implemented in the mixtools R package to derive the underlying
genetic background based on the mixture of three distributions in
the methylation levels of the DUSP22 DMR. In all samples, each
participant was allocated to one of the three “genetic”groups
based on the level of methylation. To investigate the potential
confounding influence of genotype, we used membership to the
methylation distribution (1,2,3) as ordinal indicator in χ
2
tests,
logistic regression, or linear regression as appropriate (see
supplement 1). For the stratified analysis by genotype levels
(excluding participants with membership to low methylation
genetic background), genetic background was not added to the
models.
Analysis of expression in brain and blood
Details of the analysis of the relationship between DUSP22
methylation and expression of transcript levels can be found in
the supplement. In short, we analyzed the association between
DNA methylation levels of the DUSP22 DMR and DUSP22
expression and adjusted for the underlying genetic background.
In situ Hi-C data from human postmortem brain tissue
Flash frozen postmortem brain tissue was obtained from the
Human Brain Collection Core (HBCC) of the National Institute of
Mental Health (NIMH), US. The anterior cingulate cortex sample
used in this study is from a 35-year-old non-psychiatric female.
Nuclei isolation through extraction, purification, and fluorescence-
activated nuclear sorting (FANS) was performed with minor
adjustments per Kundakovic et al.
54
(see supplement).
Nutritional deprivation of fibroblasts
Details of the procedures and analysis are available in the
supplemental materials. In short, fibroblasts from ten donors were
cultured in Minimum Essential Medium (MEM) (Gibco®) with or
without 15% FBS to mimic famine. DNA and mRNA were extracted
and analyzed using EPIC methylation arrays and qPCR. Non-
parametric analysis of average methylation of all DUSP22 CpGs for
the paired observations of all ten donors for the 72-h nutritional
deprivation compared to the condition with FBS was done using a
paired Wilcoxon Signed Rank Test.
DATA AVAILABILITY
The DNA methylation datasets generated during and/or analyzed during the curre nt
study are available in the GEO repository, https://www.ncbi.nlm.nih.gov/geo/query/
acc.cgi?acc=GSE116380
ACKNOWLEDGEMENTS
We are grateful to all participants in our study. We thank Roel de Rijk for his help with
genotyping; Ruben van het Slot and Bobby Koeleman for their help with the EPIC
arrays; Marc Bohlken and Annet van Bergen for helping with fibroblast collection.
Leonard Schalkwyk kindly provided advice regarding R coding issues. Beijing Gene-
Square Square Biotech Ltd. facilitated the methylation measurement of the Chinese
samples. Statistical analyses were carried out on the Genetic Cluster Computer
(http://www.geneticcluster.org) hosted by SURFsara and financially supported by the
Netherlands Scientific Organization (NWO 480-05-003 PI: Posthuma) along with a
supplement from the Dutch Brain Foundation and the VU University Amsterdam. This
project was facilitated by the EpiChemBio COST Action CM-1406. The funders had no
role in the design and reporting of the study.
AUTHOR CONTRIBUTIONS
M.P.B., Z.X., and R.S.K conceived the study. M.P.B., L.C.H., G.U., A.X.M., P.R., and A.E.J.
conducted the statistical analysis, Q.Y., H.X., Y.W., J.P.S., H.E.H.P., J.E.K., D.G.B., C.H.V., C.
M.N., Y.H., and J.Y. collated the data, Y.H., L.D.W., P.R., and E.S. carried out the
laboratory analysis, L.D.W., E.M.H., B.P.F.R., S.A., C.H.V., D.R.W., J.Y., and R.S.K. oversaw
the methodology, M.P.B. and L.C.H. wrote the first draft. A.E.J. provided statistical
advice. All authors contributed to and approved the final manuscript.
ADDITIONAL INFORMATION
Supplementary information accompanies the paper on the npj Schizophrenia
website (https://doi.org/10.1038/s41537-018-0058-4).
Competing interests: The authors declare no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations.
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© The Author(s) 2018
Genetic vulnerability to DUSP22 promoter hypermethylation is. . .
MP Boks et al.
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npj Schizophrenia (2018) 16 Published in partnership with the Schizophrenia International Research Society