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The World Journal of Biological Psychiatry

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Gene expression study of mitochondrial complex


I in schizophrenia and paranoid personality
disorder

Arvin Haghighatfard, Sarah Andalib, Mozhdeh Amini Faskhodi, Soha


Sadeghi, Amir Hossein Ghaderi, Shadi Moradkhani, Jalal Rostampour, Zeinab
Tabrizi, Ali Mahmoodi, Talie Karimi & Zakieh Ghadimi

To cite this article: Arvin Haghighatfard, Sarah Andalib, Mozhdeh Amini Faskhodi, Soha Sadeghi,
Amir Hossein Ghaderi, Shadi Moradkhani, Jalal Rostampour, Zeinab Tabrizi, Ali Mahmoodi,
Talie Karimi & Zakieh Ghadimi (2017): Gene expression study of mitochondrial complex I in
schizophrenia and paranoid personality disorder, The World Journal of Biological Psychiatry, DOI:
10.1080/15622975.2017.1282171

To link to this article: http://dx.doi.org/10.1080/15622975.2017.1282171

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Download by: [Fudan University] Date: 20 February 2017, At: 04:56


THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY, 2017
http://dx.doi.org/10.1080/15622975.2017.1282171

ORIGINAL INVESTIGATION

Gene expression study of mitochondrial complex I in schizophrenia and


paranoid personality disorder
Arvin Haghighatfarda, Sarah Andalibb, Mozhdeh Amini Faskhodic, Soha Sadeghid, Amir Hossein Ghaderie,
Shadi Moradkhanif, Jalal Rostampourg, Zeinab Tabrizih, Ali Mahmoodia, Talie Karimii and Zakieh Ghadimij
a
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran; bInstitute for Brain and Cognitive
Science, Shahid Beheshti University, Tehran, Iran; cDepartment of Biology, Tehran Medical Branch, Islamic Azad University, Tehran,
Iran; dLaboratory of Medical Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran; eCognitive
Neuroscience Lab, Department of Psychology, University of Tabriz, Tabriz, Iran; fDepartment of Physics, Amirkabir University of
Technology, Tehran, Iran; gDepartment of Cell & Molecular Biology, School of Biology, College of Science, University of Tehran,
Tehran, Iran; hDepartment of Medical Immunology, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran;
i
Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Iran; jDepartment of Biology, Qom
Branch, Islamic Azad University, Qom, Iran

ABSTRACT ARTICLE HISTORY


Objectives: The aetiology and molecular mechanisms of schizophrenia (SCZ) and paranoid Received 7 July 2016
personality disorder (PPD) are not yet clarified. The present study aimed to assess the role of Revised 2 December 2016
mitochondrial complex I and cell bioenergetic pathways in the aetiology and characteristics of Accepted 2 January 2017
SCZ and PPD.
Methods: mRNA levels of all genomic and mitochondrial genes which encode mitochondrial KEYWORDS
complex I subunits (44 genes) were assessed in blood in 634 SCZ, 340 PPD patients and 528 Schizophrenia; paranoid
non-psychiatric subjects using quantitative real-time PCR, and associated comprehensive psychi- personality disorder;
atric, neurological and biochemical assessments. mitochondrial complex I;
Results: Significant expression changes of 18 genes in SCZ patients and 11 genes in PPD patients gene expression; NDUFS1
were detected in mitochondrial complex I. Most of these genes were novel candidate genes for
SCZ and PPD. Several correlations between mRNA levels and severity of symptoms, drug response,
deficits in attention, working memory, executive functions and brain activities were found.
Conclusions: Deregulations of both core and supernumerary subunits of complex I are involved
in the aetiology of SCZ and PPD. These deregulations have effects on brain activity as well as
disorder characteristics.

Introduction depression and mannerisms are frequently seen in


both disorders as well. Hallucinations and delusions
Schizophrenia (SCZ) is a chronic neuropsychiatric dis-
are two common symptoms of SCZ that are not pre-
order with 1% prevalence worldwide and no certain
known aetiology. SCZ has great heterogeneity across sent in PPD patients. Several studies reported a spe-
individuals and variation in severity of positive and cific familial association between SCZ-related
negative symptoms (Ben-Shachar & Karry 2007; Clay personality disorders such as PPD, and SCZ patients
et al. 2011). Recent hypotheses have addressed both (Webb & Levinson 1993). While a series of twin studies
neurochemical and neurodevelopmental components found significant heritability for PPD in the range of
in the pathogenesis of SCZ (Dror et al. 2002; Karry 35–60%, genetic and molecular bases of PPD remain
et al. 2004; Mehler-Wex et al. 2006). unclear (Reichborn-Kjennerud 2010). As the aetiology
Paranoid personality disorder (PPD) is a mental dis- of SCZ and PPD remains unclear and their diagnosis is
order characterised by paranoia and a pervasive, long- based on clinical interviews, reliable peripheral bio-
standing suspiciousness and generalised mistrust of logical markers for these disorders are needed. Whole-
others with 0.5–2.5% occurrence in the general popu- genome gene expression profiling in SCZ revealed
lation and no clear aetiology (Waldinger 1997). gene expression alteration in several genes with differ-
Paranoid thoughts are the main shared symptoms ent functions such as neurotransmission, gene regula-
between PPD and SCZ, but hostility, social avoidance, tion, cell cycle progression, differentiation, apoptosis,

CONTACT Arvin Haghighatfard Arvinland@yahoo.com Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/15622975.2017.1282171.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
2 A. HAGHIGHATFARD ET AL.

Figure 1. Schematic representation of human complex I subunits and their putative topology within the complex. All core subu-
nits along with 30 supernumerary mammalian subunits and their positions in complex I are presented. Bovine homologues are
shown in grey and human subunits in which mutations are found are marked with an asterisk (Ugalde et al. 2004).

microRNA processing and immunity. This functional cofactors and structural elements, and are sufficient
diversity in SCZ candidate genes could be related to for catalysis. The other 30 ‘supernumerary’ subunits
pathophysiological heterogeneity of complex psychi- are mammalian specific. The supernumerary subunits
atric disorders (Sanders et al. 2013). are important in assembly, regulation, stability and
Mitochondrial complex I (NADH: ubiquinone oxidor- protection against oxidative stress. There is a lack of
eductase) has a necessary role in respiration in many high-resolution structure-limited knowledge about the
aerobic organisms. Complex I oxidise NADH from the role of 30 supernumerary mammalian subunits in the
tricarboxylic acid cycle and b-oxidation, reduces ubi- assembly and activity of complex I (Figure 1). In add-
quinone, and transports protons across the inner ition, the abnormalities or mutations in complex I sub-
membrane, contributing to the proton-motive force in units which cause human diseases are not clear
mitochondria. It is also a major contributor to cellular (Vinothkumar et al. 2014).
production of reactive oxygen species (Hirst 2013). Impaired cellular energy state, which is a conse-
Complex I, which is one of the largest and most quence of impaired mitochondrial function, is an inter-
complicated enzymes in cells, contains 44 different esting hypothesis for explaining the pathophysiology
subunits (nuclear and mitochondrial genomes) and of SCZ. An abnormal cellular energy state can lead to
nine redox cofactors. Fourteen subunits are the ‘core’ alterations in neuronal function, plasticity and brain
subunits that are conserved in complex I in all species. circuitry, and thereby to the cognitive and behavioural
Core subunits contain all the mechanistically critical aberrations characteristic of SCZ (Ben-Shachar 2002).
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 3

There is evidence which indicates that SCZ has genes in mitochondrial complex I and III in blood sam-
pathological components that could be attributed to ples of schizophrenic patients with psychiatric results
abnormalities in mitochondrial function (Mehler-Wex (Akarsu et al. 2014). They confirmed the over-expres-
et al. 2006). Also, it has been reported that mito- sion of NDUFS1, NDUFV1 and NDUFV2 and a positive
chondrial dysfunctions negatively affect neuronal correlation of NDUFV2 over-expression using the Brief
plasticity, and cause cognitive deficits, behavioural Psychiatric Rating Scale (BPRS) and simplified acute
abnormalities and executive dysfunction in several physiology score in first-episode SCZ.
neurological and neuropsychiatric disorders including Similar positive symptoms in SCZ and PPD led us to
Parkinsonism, bipolar disorder and SCZ (Akarsu et al. evaluate the same pattern of genes in both disorders
2014). to better understand the shared genetic bases of SCZ
Ben-Shachar et al. (1999) reported the enzymatic and other psychiatric and personality disorders, espe-
activity of complex I, an increase in peripheral blood cially in bioenergetic pathways. Previous studies of
of patients with SCZ. Dror et al. (2002) reported over- mitochondrial complex I activity in SCZ was focussed
activity of mitochondrial complex I in patients with in large subunits. The present study aimed to compre-
SCZ, which was associated with positive and negative hensively investigate transcript levels of all genomic
psychiatric symptoms. A study of mitochondrial and mitochondrial genes which encode mitochondrial
complex I genes has been repeated in European popu- complex I subunits, and the correlation of mRNA levels
lation by Mehler-Wex et al. (2006) and detected with psychiatric symptoms, intelligence, working mem-
over-expression of NDUFS1 in 10 schizophrenic ory, cognition, attention, executive functions, neuro-
patients (encoding greatest subunit of mitochondrial logical situation, antipsychotic treatment responses
complex I) as a marker for SCZ. Akarsu et al. (2014) and the biochemistry of SCZ and PPD in a large num-
studied the correlation between the expression of four ber of samples (Tables 1 and 2).

Table 1. Clinical characteristics in SCZ, PPD and non-psychiatric subjects.


Variables SCZ PPD Non-psychiatric
Age 31 (28–35) 33 (27–36) 30 (27–32)
Age of onset (years) 22.5 (18–27) 28.5 (19–30) –
Duration of illness (month) 39.7 (28–41) 16.4 (12–19) –
Duration of hospitalization (day) 16.6 (15–17) 15.4 (14–18) –
Estimated antipsychotic medications (mg/day) Haloperidol: 2 Haloperidol: 3 –
Clozapine: 600 Risperidone: 2
Clonazepam: 0.5 diazepam: 250
Risperidone: 2
Olanzapine: 20
Fluoxetine: 50
Number of antipsychotic resistant 41 6 –
PANSS total 74.42 (64–79) – –
General symptoms 31.32 (27–34)
Positive score 27.38 (24–32) – –
Negative score 14.76 (12–18) – –
BPRS total 56.29 (41to 59) – –
withdrawal/Retardation 6.1 (5–7) – –
Anxious/Depression 8.45 (8–9) – –
Hostile/Suspiciousness 12.19 (10–13) – –
Thinking disturbance 14.34 (13–16) – –
IQ total score 85.3 (82–106) 95.44 (93–112) 98.23 (90–118)
Neutral stimuli Stroop test score 815.9 (728–840) 763.3 (684–785) 684(634–712)
Incongruent stimuli Stroop test score 967.6 (829–983) 914.3 (781–946) 827 (775–855)
Congruent stimuli Stroop test score 852.5 (811–874) 720.8 (634–736) 682(613–707)
WSCT correct responses 59.34 (42–68) 67.32(51–77) 78.13 (73–80)
WSCT completed groups 2.1 (1–4) 2.55 (1–4) 3.86 (2–4)
TMT-A 64.23 (48–68) 52.6 (43–62) 37.4 (29–42)
TMT-B 148.56 (122–156) 111.49 (102–133) 82.36 (66–142)
SOD(U/ml) 0.11 (0.9–0.14) 0.28 (0.24–0.33) 0.35 (0.24–0.41)
EEG-ERP P300-auditory: 593 (508–602) ms P300-auditory: 521 (487–532) ms P300-auditory: 408 (390–432) ms
P300-visual: 542 (494–563) ms P300-visual: 515 (502–568) ms P300-visual: 411 (367–422) ms
MMN standard tone: MMN standard tone: 1881 (1824 to1897) MMN standard tone:
1893 (1811–1924) MMN frequency deviant: 203 (196–211) 1729 (1683–1836)
MMN frequency deviant: MMN duration deviant: 198.4 (182–205) MMN frequency deviant:
223 (206–234) 196.4 (188–202)
MMN duration deviant: MMN duration deviant:
201 (192–207) 197 (191–204)
PANSS: Positive and Negative Syndrome Scale; BPRS: Brief Psychiatric Rating Scale; WSCT: Wisconsin Card Sorting Test; TMT: Trail Making Test; SOD: super-
oxide dismutase; EEG-ERP: electroencephalography–event-related potential; MMN: mismatch negativity.
4 A. HAGHIGHATFARD ET AL.

Table 2. Gene mRNA levels of the cases; ratio (mean ratio of gene mRNA levels in patient groups in comparison with non-psy-
chiatric subjects) and statistical results (group comparisons for mRNA levels of 44 genes of interest versus four housekeeping
genes, due to 0.05/44 ¼ 0.001042)
SCZ vs PPD vs Paranoid SCZ vs
non-psychiatric non-psychiatric SCZ vs non-psychiatric other SCZ patients PPD vs non-psychiatric
Genes Subunits subjects (Ratio) subjects (Ratio) subjects (P and F values) (P and F values) subjects (P and F values)
MT-ND1 core subunit 1 0.98 ± 0.66 0.96 ± 0.72 P¼0.12 P¼0.72 P¼0.24
F¼2.21 F¼1.08 F¼1.44
MT-ND2 core subunit 2 1.02 ± 0.23 0.92 ± 0.41 P¼0.28 P¼0.54 P¼0.16
F¼1.32 F¼1.09 F¼2.11
MT-ND3 core subunit 3 0.95 ± 0.46 0.93 ± 0.51 P¼0.58 P¼0.88 P¼0.74
F¼1.19 F¼0.78 F¼0.68
MT-ND4 core subunit 4 0.83 ± 0.29 1.08 ± 0.27 P¼0.35 P¼0.67 P¼0.56
F¼1.64 F¼0.87 F¼1.53
MT-ND4L core subunit 4L 1.12 ± 0.34 1.03 ± 0.55 P¼0.3 P¼0.94 P¼0.56
F¼1.55 F¼0.61 F¼1.03
MT-ND5 core subunit 5 0.92 ± 0.4 0.87 ± 0.18 P¼0.43 P¼0.82 P¼0.62
F¼1.13 F¼0.38 F¼0.94
MT-ND6 core subunit 6 0.88 ± 0.25 1.06 ± 0.19 P¼0.22 P¼0.46 P¼0.29
F¼1.83 F¼1.76 F¼2.02
NDUFA1 supernumerary A1 0.63 ± 0.35 0.87 ± 0.48 P¼0.0002 P¼0.23 P¼0.11
F¼21.18 F¼2.17 F¼2.24
*down-regulated
NDUFA2 supernumerary A2 0.58 ± 0.62 0.8 ± 0.28 P¼0.0003 P¼0.34 P¼0.08
F¼19. 26 F¼1.13 F¼7.12
*down-regulated
NDUFA3 supernumerary A3 0.85 ± 0.36 0.91 ± 0.67 P¼0.51 P¼0.84 P¼0.72
F¼2.17 F¼1.35 F¼1.27
NDUFA5 supernumerary A5 1.44 ± 0.27 1.32 ± 0.53 P¼0.0004 P¼0.13 P¼0.0004
F¼16.76 F¼2.13 F¼18.21
*up-regulated *up-regulated
NDUFA6 supernumerary A6 0.98 ± 0.04 0.94.± 0.11 P¼0.32 P¼0.4 P¼0.41
F¼1.24 F¼2.11 F¼1.33
NDUFA7 supernumerary A7 0.96 ± 0.24 0.89 ± 0.57 P¼0.6 P¼1.2 P¼0.81
F¼1.19 F¼0.27 F¼0.17

NDUFA8 supernumerary A8 1.74 ± 0.79 1.67 ± 0.26 P¼0.0008 P¼0.18 P¼0.0006


F¼14.66 F¼2.94 F¼17.74
*up-regulated *up-regulated
NDUFA9 supernumerary A9 1.07 ± 0.47 1.05 ± 0.39 P¼0.65 P¼0.52 P¼0.39
F¼0. 82 F¼0.8 F¼1.54
NDUFA10 supernumerary A10 0.85 ± 0.17 0.95 ± 0.42 P¼0.32 P¼0.69 P¼0.44
F¼1.15 F¼0. 66 F¼1.05

NDUFA11 supernumerary A11 1.06 ± 0.24 0.99 ± 0.33 P¼0.8 P¼0.65 P¼0.73
F¼0.32 F¼0.93 F¼0.79

NDUFA12 supernumerary A12 0.94 ± 0.11 1.03 ± 0.35 P¼0.31 P¼0.44 P¼0.26
F¼2.04 F¼1.38 F¼1.54

NDUFA13 supernumerary A13 1.69 ± 0.55 1.73 ± 0.49 P¼0.0008 P¼0.29 P¼0.0005
F¼18.36 F¼2.24 F¼16.19
*up-regulated *up-regulated
NDUFAB1 supernumerary AB1 0.47 ± 0.23 0.87 ± 0.31 P¼0.0004 P¼0.54 P¼0.37
F¼15.71 F¼0.86 F¼1.32
*down-regulated
NDUFB1 supernumerary B1 0.95 ± 0.24 0.93 ± 0.54 P¼0.58 P¼0.48 P¼0.34
F¼1.22 F¼1.32 F¼1.46

NDUFB2 supernumerary B2 1.48 ± 0.37 0.86 ± 0.22 P¼0.0002 P¼0.56 P¼0.42


F¼21.17 F¼1.04 F¼1.28
*up-regulated
NDUFB3 supernumerary B3 1.02 ± 0.56 0.97 ± 0.36 P¼0.21 P¼0.72 P¼1.06
F¼1.33 F¼0.49 F¼0.08

NDUFB4 supernumerary B4 1.1 ± 0.26 0.95 ± 0.28 P¼0.38 P¼0.57 P¼0.49


F¼1.28 F¼0.92 F¼1.19

NDUFB5 supernumerary B5 1.58 ± 0.62 1.52 ± 0.34 P¼0.0003 P¼0.17 P¼0.0002


F¼18.23 F¼3.05 F¼19.16
*up-regulated *up-regulated
NDUFB6 supernumerary B6 0.95 ± 0.46 0.9 ± 0.31 P¼0.52 P¼0.78 P¼0.44
F¼2.14 F¼1.55 F¼1.65
(continued)
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 5

Table 2. Continued
SCZ vs PPD vs Paranoid SCZ vs
non-psychiatric non-psychiatric SCZ vs non-psychiatric other SCZ patients PPD vs non-psychiatric
Genes Subunits subjects (Ratio) subjects (Ratio) subjects (P and F values) (P and F values) subjects (P and F values)

NDUFB7 supernumerary B7 1.12 ± 0.18 1.01 ± 0.53 P¼0.27 P¼0.54 P¼0.46


F¼3.12 F¼1. 6 F¼1.95

NDUFB8 supernumerary B8 1.03 ± 0.39 1.09 ± 0.24 P¼0.38 P¼1.19 P¼0.24


F¼2.2 F¼0.45 F¼2.39

NDUFB9 supernumerary B9 1.68 ± 0.46 1.79 ± 0.61 P¼0.0007 P¼0.86 P¼0.0004


F¼17.23 F¼0.13 F¼19.1
*up-regulated *up-regulated
NDUFB10 supernumerary B10 0.48 ± 0.78 0.84 ± 0.52 P¼0.0003 P¼0.83 P¼0.04
F¼20.14 F¼0.9 F¼7.06
*down-regulated
NDUFB11 supernumerary B11 0.56 ± 0.42 0.78 ± 0.59 P¼0.0008 P¼0.18 P¼0.0003
F¼16.24 F¼2.43 F¼19.38
*down-regulated *down-regulated
NDUFC1 supernumerary C1 1.54 ± 0.53 1.13 ± 0.16 P¼0.0004 P¼0.79 P¼0.06
F¼18.21 F¼1.17 F¼5.28
*down-regulated
NDUFC2 supernumerary C2 1.05 ± 0.36 1.08 ± 0.54 P¼1.02 P¼0.51 P¼0.89
F¼0.94 F¼1.63 F¼1.03

NDUFS1 core subunit S1 1.89 ± 0.73 1.74 ± 0.28 P¼0.0001 P¼0.0006 P¼0.0002
F¼22.36 F¼17.36 F¼19.84
*up-regulated *up-regulated *up-regulated
NDUFS2 core subunit S2 0.84 ± 0.7 0.96 ± 0.65 P¼0.87 P¼0.59 P¼0.86
F¼1.0 F¼1.94 F¼1.02

NDUFS3 core subunit S3 1.04 ± 0.58 0.94 ± 0.66 P¼0.24 P¼0.71 P¼0.9
F¼2.15 F¼1.3 F¼0.58

NDUFS4 supernumerary S4 1.62 ± 0.46 1.05 ± 0.74 P¼0.0005 P¼0.83 P¼0.85


F¼17.32 F¼1.12 F¼1.01
*up-regulated

NDUFS5 supernumerary S5 1.2 ± 0.4 0.97 ± 0.86 P¼0.52 P¼0.49 P¼0.73


F¼1.21 F¼1.53 F¼0.96

NDUFS6 supernumerary S6 0.96 ± 0.55 1.03 ± 0.37 P¼0.09 P¼0.64 P¼0.12


F¼4.27 F¼1.12 F¼3.08

NDUFS7 core subunit S7 0.75 ± 0.54 0.68 ± 0.66 P¼0.0008 P¼0.44 P¼0.0003
F¼16.21 F¼2.2 F¼18.42
*down-regulated *down-regulated
NDUFS8 core subunit S8 0.69 ± 0.58 0.54 ± 0.62 P¼0.0006 P¼0.72 P¼0.0007
F¼15.57 F¼1.24 F¼13.09
*down-regulated *down-regulated
NDUFV1 core subunit V1 1.67 ± 0.8 1.71 ± 0.65 P¼0.0004 P¼0.09 P¼0.0006
F¼16.18 F¼2.32 F¼15.63
*up-regulated *up-regulated
NDUFV2 core subunit V2 1.85 ± 0.63 1.73 ± 0.78 P¼0.0007 P¼0.12 P¼0.0002
F¼16.22 F¼2.07 F¼18.3
*up-regulated *up-regulated
NDUFV3 supernumerary V3 1 ± 0.32 1.04 ± 0.73 P¼0.67 P¼0.74 P¼0.35
F¼1.12 F¼1.14 F¼2.17
Significant changes/non-significant changes.

Materials and methods medications, in the 18–24-month period before sam-


pling and were considered as chronic patients.
Subject selection
Patients were selected from psychiatric hospitals and
The study included 634 unrelated SCZ patients clinics of 22 out of the 31 provinces of Iran. Patients
(414 male, 220 female) and 340 unrelated PPD patients with schizoaffective or schizotypal illness were
(204 male, 136 female), diagnosed by two independ- excluded. Chlorpromazine (CPZ) equivalents used to
ent senior psychiatrists based on extended clinical give the estimated average daily dose; and estimated
interview and patients’ DSM-V chart review. All of the lifetime CPZ equivalents considering of duration of
patients had started treatment, including antipsychotic illness. The non-psychiatric group included 528
6 A. HAGHIGHATFARD ET AL.

non-psychiatric subjects (325 male, 203 female) with The Stroop test is a neuropsychological test which
matched sex, age, race, socioeconomic situation, famil- is used to measure sustained attention. This test is
ial situation and education to the patient groups (non- based on the Stroop effect, which is a demonstration
significant P values in the basic demographic situation of interference in the reaction time of a task and using
are presented in Supplementary Table 1, available the name of a colour which is printed in a colour not
online) and with no history of any psychological prob- denoted by the name (Carter et al. 1997).
lems, no current or history of severe medical condi- The Trail Making Test is a neuropsychological test
tions, neurological disorder, history of head trauma of visual attention and task switching. It is also use to
with loss of consciousness, no psycho-stimulant or opi- examine cognitive and executive functioning
oid drug abuse and alcohol or nicotine dependence. (Gonzalez-Liencres et al. 2014).
Subjects were given an explanation on the purpose of
the study and then written informed consent was pro-
Neurological assessment
vided according to the Declaration of Helsinki. The
study was approved by central ethical committee of Electroencephalography (EEG) was recorded using cus-
Islamic Azad University in Tehran. tom-designed electrode caps with a 64-channel
BioSemi Active-Two system (BioSemi BV) and data were
processed by the Brain Vision Analyser package, version
Analysis of clinical data and psychiatric
2.0 (Brain Products GmbH, Munich, Germany). Three
assessment
event-related potentials were measured: mismatch
Clinical information including duration of illness, sub- negativity (MMN), amplitude of P300 auditory oddball
types of SCZ (297 paranoid, 33 catatonic, 53 disorgan- task, and P300 visual amplitude. The methods and data
ised, 168 undifferentiated and 83 residual), analysis processes were operated according to previous
antipsychotic resistance and familial history of psych- studies (Silver et al. 2003; Del Re et al. 2014).
osis were extracted from patient's files. Two trained
senior psychiatrists performed the ratings independ-
Blood sampling and real-time PCR
ently and a consensus rating was obtained.
Symptom severity was measured with the Positive Blood samples(15 ml) were collected from the cubital
and Negative Syndrome Scale (PANSS), a 30-item semi- vein without tourniquet using PAXgene blood RNA
structured interview designed to assess three symp- tubes (Cat No762165) between 08.00 and 09.00 h in
tom categories associated with SCZ: positive, negative the hospital laboratory sampling rooms and RNA
and general symptoms (APA 1994). extraction started immediately after sampling. Total
The 24-item version of the BPRS is a rating scale for RNA was extracted from peripheral blood samples
psychiatric symptoms such as depression, anxiety, hal- according to the standard protocols of the RNA
lucinations and unusual behaviour (Ventura et al. Purification kit (GeneJETTM RNA Purification Kit#K0732,
2000). Fermentas, Latvia). Total RNA was treated with DNase
for the removal of contaminating genomic DNA using
DNase Treatment & Removal Reagents (DNase I,
Neuropsychological assessment
RNase-free (#EN0521) Fermentas, Latvia), according to
The Wisconsin Card Sorting Test (WCST) is a neuro- the manufacturers protocol. The quality and integrity
psychological test of ‘set-shifting’, which use to assess of extracted RNA was examined by gel electrophoresis
the following ‘frontal’ lobe functions and study of and UV spectroscopy, and sampling repeated for sub-
executive functions (Drake & Lewis 2003). A number of jects with low-quality RNA in the first sample.
stimulus cards are presented to the participant. The The cDNA was synthesised with a Transcription First
participant is told to match the cards, but not how to Strand cDNA Synthesis Kit (RevertAid Premium First
match; but the participant is told whether a particular Strand cDNA Synthesis Kit #K1652, Fermentas, Latvia)
match is right or wrong (Kaufman & Lichtenberger according to the manufacturer’s protocol. Primers for
2005). all genes designed by ‘Gene runner version 3.05’ and
The Wechsler Adult Intelligence Scale (WAIS) is a ‘oligo7’ software and blasted on the NCBI website (pri-
test designed to measure intelligence in adults and mer sequences are presented in Supplementary Table
older adolescents. Digit span and Dot span are two 2). An average of four housekeeping genes (b-actin,
subtests of WAIS which examine the verbal and PGK1, EMC7 and CHMP2A) were used for normalisation
Spatial working memory (Kaufman & Lichtenberger as endogenous reference genes (CT values of house-
2005). keeping genes in all subjects mean ± SD are presented
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 7

in Supplementary Table 3). PCR and agarose gel elec- Compliance with normal distribution for continuous
trophoresis were used to verify the predicted size of variables was assessed via the Kolmogorov-Smirnov
PCR amplicons for all 48 genes. Standard curves for test. For multiple group comparisons, statistical differ-
each gene were prepared using serial dilutions (1:4) of ences were calculated by one-way ANOVA followed by
pooled cDNA from total RNA extracted from blood independent Student’s t-test. Pearson correlation ana-
samples of 10 non-psychiatric subjects. In each experi- lysis was conducted to determine the relationship
ment, the R2 value of the standard curve was more between variables. Bonferroni correction was used for
than 0.99, and no-template control assays resulted in multiple comparisons corrections. To control of any
no detectable signal. Quantitative real-time PCR (qPCR) potential confounds, body mass index (BMI), medica-
was performed using syber green (Thermo Scientific tion classes, RNA quality and concentration, cDNA syn-
Maxima SYBR Green/ROX qPCR Master Mix (2X) thesis quality, plates/runs of qPCR and primer
#K0221, Fermentas, Latvia). A triplicate method was efficiency were added as covariates and persistence of
performed for quantitative real-time PCR using a the significant difference in main effect between
7900HT Fast Real-Time PCR System with a Fast 96-Well groups was assessed by ANCOVA. Statistical analysis
Block Module (Applied Biosystems, Foster City, CA, conducted by using SPSS version23.
USA). PCR data were obtained by Sequence Detector
Software (SDS version 2.3 Rev C Patch, Applied
Results
Biosystems) and quantified by the standard curve
method. This software plotted the real-time fluores- The clinical and demographic data of the non-psy-
cence intensity and selected the threshold within the chiatric subjects and patients, including medications,
linear phase of the amplicon profile. The software plot- are given in parts A and B of Table 1. ANCOVA
ted a standard curve of the cycle at threshold versus examinations showed BMI (P ¼ 0.45, f ¼ 0.32), medi-
extracted RNA quantity. Samples were measured in cation classes (P ¼ 0.27, f ¼ 2.04), RNA quality and
one plate for one target gene and their Ct values were concentration (P ¼ 0.23, f ¼ 2.13), cDNA synthesis
in the linear range of the standard curve. In qPCR quality (P ¼ 0.39, f ¼ 1.08), plates/runs of qPCR
tests, outliers or sample failures were repeated for (P ¼ 0.21, f ¼ 2.21) and primer efficiency (P ¼ 0.42,
each gene. The ratio was calculated using the pfafle f ¼0.87), which were added as covariates and had no
formula. effect on the statistical significance of the differences
between groups.

Biochemical assessment
Gene expression results
Several studies suggest that oxidative stress might
serve as a potential biomarker in the aetiology and In present study, expression changes of 18 genes in
clinical course of SCZ (Oribe et al. 2014). Subjects were SCZ patients and 11 genes in PPD patients were
asked to sleep normally the previous night and refrain detected in mitochondrial complex I. Expression levels
from eating, using drugs, drinking alcohol or doing of NDUFS1, NDUFS4, NDUFV1, NDUFV2, NDUFB2,
physical exercise 8 h before the sampling. All persons NDUFB5, NDUFB9, NDUFA5, NDUFA8, NDUFA13 and
with any smoking history were excluded from the NDUFC1 were up-regulated and expression levels of
study. Blood samples were collected from the partici- NDUFAB1, NDUFB10, NDUFB11, NDUFA1, NDUFA2,
pants at admission time (08:00 to 09:00 h). Plasma and NDUFS7 and NDUFS8 were down-regulated in chronic
serum were obtained by centrifugation and, together schizophrenic patients in comparison with non-psychi-
with the whole-blood sample, were kept at 80  C atric individuals. Also, expression levels of NDUFS1,
until analysis. Superoxide dismutase (SOD) was ana- NDUFV1, NDUFV2, NDUFB5, NDUFB9, NDUFA13, NDUFA8
lysed from serum. SOD activity was determined using and NDUFA5 were up-regulated and NDUFB11, NDUFS7
enzyme-linked immunosorbent assay, which measures and NDUFS8 were down-regulated in the PPD group
the three types of SOD (Cu/Zn, Mn and Fe), according in comparison with the non-psychiatric group.
to the manufacturer’s instructions (Cayman Chemical Significant over-expression of NDUFS1 was found in
Company, MI, USA). Absorbance was read at 450 nm. paranoid schizophrenic (n ¼ 297) and PPD patients
(n ¼ 340) in comparison with other subtypes of
SCZ (total n ¼ 337). Gene expression results are pre-
Statistical analysis sented in Table 2 and Figures 2 and 3, which show a
Descriptive data are expressed as mean ± SD (range) heat map of gene expression levels in group
and level of statistical significance was set at P < 0.05. comparisons.
8 A. HAGHIGHATFARD ET AL.

Figure 2. Volcano plot visualisation of gene expression level differences in SCZ versus non-psychiatric groups. From 44 genes, sig-
nificantly over-expressed genes (NDUFS1, NDUFS4, NDUFV1, NDUFV2, NDUFB2, NDUFB5, NDUFB9, NDUFA5, NDUFA8, NDUFA13
and NDUFC1) are illustrated in the top left of the plot, and significantly under-expressed genes (NDUFAB1, NDUFB10, NDUFB11,
NDUFA1, NDUFA2, NDUFS7 and NDUFS8) are illustrated on the top right of the plot. Genes with no significant alteration are illus-
trated under the horizontal line.

Clinical characteristics (P ¼ 0.0009), NDUFB9 (P ¼ 0.0008), NDUFV1 (P ¼ 0.0006)


and NDUFV2 (P ¼ 0.0009) and under-expression of
A summary of all the different correlation data, including
NDUFB11 (P ¼ 0.0004) and NDUFA1 (P ¼ 0.0006) with
correlation coefficient r values for each gene, is provided
total scores of PANSS and BPRS in schizophrenic
in Supplementary Table 3 (sheet 1 for SCZ and sheet 2
patients. In addition, a direct correlation was found
for PPD). In addition, two heat maps presented in
between positive symptom scores and NDUFS1
Figures 4 and 5 illustrate all the correlations between
gene expression levels and demographic and clinical (P ¼ 0.0006) and NDUFB9 (P ¼ 0.0009) expression, and a
data in SCZ and PPD, respectively. Because of the poten- direct correlation between negative symptom scores
tial effect of age on gene mRNA levels, patients of a par- and NDUFB11 (P ¼ 0.001) and NDUFA1 (P ¼ 0.0007) and
ticular age range were selected. No significant correlation under-expression in PANSS. In BPRS subscales, with-
between expression results of any genes with gender, drawal/Retardation and Anxious/Depression scores
age of onset and antipsychotic dosages were observed were positively correlated to under-expression of
in the two patient groups. Duration of illness in the SCZ NDUFB11 (P ¼ 0.0008) and NDUFA1 (P ¼ 0.001), and
group was positively correlated with over-expression of Hostile/Suspiciousness score was positively correlated
NDUFS1 (P ¼ 0.0007) or NDUFV1 (P ¼ 0.0009), and under- to over-expression of NDUFS1 (P ¼ 0.0002) and NDUFB9
expression of NDUFA1 (P ¼ 0.0007). There was a direct (P ¼ 0.001). No significant correlation observed for
correlation between antipsychotic resistance in patients Thinking Disturbance score.
(41 subjects listed as drug resistant in SCZ and six sub-
jects in PPD) and over-expression of NDUFS1 (P ¼ 0.0005) Neuropsychological tests results
and NDUFV1 (P ¼ 0.0008) and under-expression of
NDUFB11 (P ¼ 0.0004) and NDUFA1 (P ¼ 0.0003). A significant deficit in working memory, attention, cog-
nition and executive functions was detected in SCZ
and PPD groups in comparison with non-psychiatric
Psychiatric tests results
subjects. There was a significant negatively correlation
There was a significant positive correlation between between over-expression of NDUFV2 (P ¼ 0.0004),
over-expression of NDUFS1 (P ¼ 0.0002), NDUFS4 NDUFB9 (P ¼ 0.001) and NDUFS1 (P ¼ 0.0004) and
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 9

Figure 3. Volcano plot visualisation of gene expression level differences in PPD versus non-psychiatric groups. From 44 genes, sig-
nificantly over-expressed genes (NDUFS1, NDUFV1, NDUFV2, NDUFB5, NDUFB9, NDUFA13, NDUFA8 and NDUFA5) are illustrated on
the top left of the plot, and significantly under-expressed genes (NDUFB11, NDUFS7 and NDUFS8) are illustrated on the top right
of the plot. Genes with no significant alteration are illustrated under the horizontal line.

under-expression of NDUFA1 (P ¼ 0.001) and WCST cor- amplitude in SCZ and PPD cases in comparison with
rect responses in both patient groups.No significant non-psychiatric subjects. Also, a deficit in MMN ampli-
correlation was observed for the number of completed tude was revealed in SCZ patients. There was a correl-
groups. No significant correlation was observed for ation between p300 visual latency in the task
total IQ score in WAIS-IV, but there were correlations performance and NDUFS1 (P ¼ 0.001) and NDUFB9
with digit and dot span subtests. Negative correlations (P ¼ 0.0006) over-expression and NDUFAB11
between under-expression of NDUFA1 (P ¼ 0.001), (P ¼ 0.0008) under-expression in SCZ and PPD groups.
NDUFB10 (P ¼ 0.001) and NDUFB11 (P ¼ 0.001) and digit There was a significant correlation between MMN def-
span forward scores and negative correlations icit (in standard tone, duration and frequency deviant)
between under-expression of NDUFA1 (P ¼ 0.0005) and with NDUFS1 (P ¼ 0.0009), NDUFV1 (P ¼ 0.001), NDUFV2
NDUFB11 (P ¼ 0.001) and over-expression of NDUFS1 (P ¼ 0.001) and NDUFB9 (P ¼ 0.001) over-expression
(P ¼ 0.0008) with digit span backward scores observed and NDUFA1 (P ¼ 0.001) and NDUFB11 (P ¼ 0.0007)
in schizophrenic and PPD patients. No significant cor- under-expression observed in both patient groups. No
relation was observed between gene expressions and correlation was observed in the p300 auditory oddball
dot span scores in patients. There was a significant task abnormalities and gene expression.
positive correlation between congruent stimuli score
and under-expression of NDUFA1 (P ¼ 0.001) and
Biochemical test results
NDUFB11 (P ¼ 0.0009) in the Stroop test in SCZ and
PPD. In the TMT test there was a negative correlation A significant reduction of the three types of SOD (Cu/
between under-expression of NDUFB11 (P ¼ 0.001) and Zn, Mn and Fe) was detected in SCZ and PPD groups
TMT-A and TMT-B second results in SCZ and PPD in comparison with non-psychiatric subjects. SOD
groups. reduction in SCZ was reported in previous studies
(Potluri et al. 2009; Gonzalez-Liencres et al. 2014), but
this is the first report of SOD reduction in PPD
Neurological tests results
patients. A significant correlation between SOD reduc-
In the auditory oddball task and visual p300 we tion and positive symptoms in PANSS was revealed in
observed a latency and significant reduction in SCZ patients. There was a significant correlation
10 A. HAGHIGHATFARD ET AL.

Figure 4. Heat map visualisation of R values between gene expression levels versus demographic data and clinical tests scores in
schizophrenic patients. Black arrays show lower R values, while brighter arrays correspond to higher R values. Green represents an
indirect relation and red represents a direct relation.

between NDUFS1 (P ¼ 0.0003), NDUFV1 (P ¼ 0.0005), small subunits as well. This is the first gene expression
NDUFV2 (P ¼ 0.001), NDUFB9 (P ¼ 0.001) and NDUFC1 study in PPD. Shared gene expression patterns of both
(P ¼ 0.001) over-expression and reduction of SOD in disorders support the hypothesis of a similarity in
the two patient groups. pathophysiology of PPD and SCZ. Eighteen candidate
genes were detected in SCZ and 11 genes in PPD; all
of the candidate genes found in PPD were among the
Discussion 18 differentially expressed genes found in SCZ and
their alteration directions were the same in both disor-
Novel marker genes in SCZ, paranoid subtype and
ders. These findings confirmed role of genomic core
PPD
subunits in SCZ. Down-regulation of NDUFS7 and
Gene expression studies of mitochondrial complex I NDUFS8 along with up-regulation of NDUFS1, NDUFV1
help to clarify the pattern of bioenergetics effects in and NDUFV2 were detected in both SCZ and PPD
behaviour and psychiatric disorders. Several studies patients. Deregulation of these five subunits from 14
which considered the gene expression of the largest core subunits may cause abnormal structured complex
subunits of mitochondrial complex I presented it as I in neural cells, which leads to abnormal energetic
potential peripheral biomarker for SCZ (Taurines et al. state and altered neural activity. None of the seven
2010; Akarsu et al. 2014). The present study examined mitochondrial genes showed a change in expression,
gene expression of whole subunits in complex I in two which may reduce the importance of a maternal pat-
psychiatric disorders for the first time, and detected a tern in the heritance of SCZ and PPD. Over-expression
pattern of gene expression alterations including up- of several supernumerary subunits was detected in
and down-regulations showing the importance of SCZ and PPD patients. As oxidative stress is increased
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 11

Figure 5. Heat map visualisation of R values between gene expression levels versus demographic data and clinical tests scores in
PPD patients. Black arrays show lower R value, while brighter arrays correspond to higher R values. Green represents an indirect
relation and red represents a direct relation.

in psychiatric disorders, it may relate to the role of in the molecular mechanisms of paranoid behaviour.
supernumerary subunits in protection against oxidative Medication effects on gene expression are one of the
stress. Under-expression of five supernumerary subu- most important obstacles which make it hard to inter-
nits was detected in SCZ. Under-expression of pret whether alterations of the mRNA levels of a gene
NDUFB11 (which is only under-expressed in super- are related to the disease rather than the medications.
numerary subunits in PPD patients) was reported in Also, complex I genes are not the direct target of anti-
brain tissue samples from SCZ patients (Huang et al. psychotics, and the significant expression changes in
2014). Also, down-regulation of the NDUFA1 gene was genes in this study are stronger than that could be
reported in progressive neurodegenerative diseases, related to antipsychotic usage. In addition, there was
including Alzheimer’s disease (Fernandez-Moreira et al. no relation between medication classes and gene
2007). expression that would reduce the importance of medi-
The results reveal NDUFS1 as a specific marker for cation in complex I gene expression.
the paranoid subtype of SCZ with significant over-
expression in comparison with other subtypes. These
Antipsychotic resistance was associated with gene
results may introduce NDUFS1 as the first subtype-
expression
specific biomarker in SCZ and revive the hypothesis
about the reclassification of SCZ and the separation of The target pathways of antipsychotics are dopamin-
the paranoid subtype from SCZ. Over-expression of ergic and serotonergic. Previous studies showed that
NDUFS1 in PPD patients alos supports this hypothesis. mitochondrial function is modulated by dopamine
Significant over-expression of NDUFS1 in PPD and the inhibition of respiratory complex I activity (Huang
paranoid subtype could reveal it as a candidate gene et al. 2014). Therefore, it is not surprising that gene
12 A. HAGHIGHATFARD ET AL.

expression alternation in mitochondrial complexes The correlation of attention deficit with NDUFA1 and
affects pharmacodynamic responses. Regardless of the NDUFB11 under-expression may suggest the potential
limited sample size, the correlation of NDUFS1, effect of complex I activity in symptoms of attention
NDUFV1, NDUFB11, and NDUFA1 mRNA levels with disorders, such as SCZ or ADHD. The correlation of
antipsychotic resistance could be related to neurode- TMT results with the under-expression of NDUFB11
generation of dopaminergic neurons due to the over- could imply a role of this gene in cognition deficiency
activity of complex I. in SCZ, PPD and other cognitive disorders like delirium,
dementia and amnesia.
Association of clinical symptoms with gene
expression changes in SCZ EEG-ERP abnormalities are related to gene
The correlations between total score of BPRS and expression changes
PANSS and mRNA levels of genes indicated a role of MMN, P300 visual and auditory abnormalities have
complex I in the behavioural pathology of SCZ. been reported in SCZ (Frantseva et al. 2014).
Correlation of the positive symptoms score of PANSS, Neurological abnormalities could indicate that endo-
and the Hostile/Suspiciousness score in BPRS, with phenotypes are involved in psychiatric disorders.
over-expression of NDUFS1 and NDUFB9 genes indi- Correlation of genetic variants or expression patterns
cates that these genes are strong markers for paranoid with neurological aberrants in SCZ and PPD could clar-
thoughts, paraphrenia and delusional disorder. Also, ify the role of the genes in neural functions as well as
correlations between the negative symptoms score of in disorders. MMN and WSCT findings, and digit span
PANSS, the withdrawal/Retardation score and the results, indicate a deficit in prefrontal function which is
Anxious/Depression score of BPRS with the down-
related to gene expression changes, especially in
regulation of NDUFB11 and NDUFA1 suggest these
NDUFS1 and NDUFB9. P300 visual were associated
genes as potential markers for reduced social engage-
with the same gene expression pattern (NDUFS1,
ment, emotional expression and lack of motivation.
NDUFB9 and NDUFAB11) as well, which implies the
pleiotropic role of complex I genes on a number of
Alteration of the expression of complex I genes neural functions at the same time (Del Re et al. 2014).
induces neuropsychological changes Correlation of complex I genes and ERP abnormalities
could support previous reports about behavioural
Previous twin studies mentioned executive functions
as most heritable psychological traits (Friedman et al. symptoms, abnormal neuronal transmission and synap-
2008). Correlation of executive functions deficit tic plasticity in subjects with over-activated mitochon-
(showed as a WCST result) and NDUFV2, NDUFS1, drial complexes (Del Re et al. 2014).
NDUFB9 and NDUFA1 expression shows an influence
of complex I activity on the executive functions of SCZ Association of SOD reduction with gene
and PPD patients. They also revealed effects of com- expression and positive symptoms
plex I activity in frontal lobe functions as the main
region of executive functions. There is a supported SOD reduction is a marker for neurodegeneration in
hypothesis that working memory is a core deficit in several disorders including SCZ (Halliwell 1992;
SCZ; and, at a component level, ROBO1 is the only Craddock et al. 2007). The correlation between SOD
related gene for the phonological loop function of reduction and over-expression of genes that were cor-
working memory (Bates et al. 2011). Results have related with positive symptoms in PANSS and BPRS
shown correlations between NDUFA1, NDUFB10, strongly supports the behavioural effects of SOD
NDUFB11 and NDUFS1 expression changes and a lack reduction, and could reveal the mechanism of these
of function in working memory, especially in verbal effects in SCZ and PPD, which may relate to complex I
working memory. The association of memory deficit activity. The SOD reduction detected in PPD patients
and NDUFA1, NDUFB10 and NDUFB11 down-regula- could reveal more details about the pathophysiology
tion could be related to electron transport chain prob- of the disorder. Mitochondrial complex deficiency is
lems in the prefrontal cortex of SCZ and PPD patients. considered to be a cause of neurodegeneration in SCZ
This suggests the involvement of the energetic state (Konradi et al. 2004; Washizuka et al. 2006; Clay et al.
of neurons on memory situation and memory disor- 2011; Park & Park 2012), and these results show that it
ders. Attention deficits are a prominent aspect of may be considered as a marker in PPD as well. Several
cognitive dysfunction in SCZ (Carter et al. 1997). similar biological, neurological and psychological bases
THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 13

of the two different psychiatric disorders, SCZ and East and South West of the country. The study included
PPD, were determined in the present study. Iranian patients from the Persian majority and different
minority tribes such as Arabs, Kurds and Armenians.
Limitation and strengths of study
Peripheral blood transcript levels may not accurately
Acknowledgements
represent expression alterations in the brain, and a We thank the Presidency of the Young Researchers and
number of studies reported tissue-specific expression Elites Club, Prof. Karim Zaree and former Vice-Presidency of
of mitochondrial complex I genes, which is the main Research and Technology in Islamic Azad University, Science
and Research Branch, and Professor Mansor Bayat for all of
limitation of the present study (Wirtz et al. 2011).
their support. This study is supported by the Young
However, as the genetic background of the subjects is Researchers and Elites Club under annual grants of special
not clear, potential genetic confounding between talents members.
cases and controls may affect the results. The present
study also has several strengths. The large sample size
increases the validity of data analysis. In SCZ, this is Disclosure statement
the first gene expression study of whole complex I The authors report no conflicts of interest. The authors alone
genes which showed a role of supernumerary subu- are responsible for the content and writing of this article.
nits, and newly assessed, core subunits in the aeti-
ology of SCZ, and completed the partial patterns that Funding
had been reported in previous studies. This is the first
This study is supported by the Young Researchers and Elites
molecular genetics study on PPD patients that high-
Club under annual grants of special talents members.
lights the molecular mechanisms, especially in the bio-
energetic pathways, in the aetiology of this personality
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