Mitochondrial DNA copy number of peripheral blood in bipolar disorder: The present study and a meta-analysis

Accepted Manuscript

Mitochondrial DNA copy number of peripheral blood in bipolar disorder: the present study and a meta-analysis Naruhisa Yamaki , Ikuo Otsuka , Shusuke Numata , Masaya Yanagi , Kentaro Mouri , Satoshi Okazaki , Shuken Boku , Tadasu Horai , Tetsuro Ohmori , Osamu Shirakawa , Ichiro Sora , Akitoyo Hishimoto PII: DOI: Reference:

S0165-1781(18)30593-6 https://doi.org/10.1016/j.psychres.2018.08.014 PSY 11608

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Psychiatry Research

Received date: Revised date: Accepted date:

2 April 2018 20 July 2018 9 August 2018

Please cite this article as: Naruhisa Yamaki , Ikuo Otsuka , Shusuke Numata , Masaya Yanagi , Kentaro Mouri , Satoshi Okazaki , Shuken Boku , Tadasu Horai , Tetsuro Ohmori , Osamu Shirakawa , Ichiro Sora , Akitoyo Hishimoto , Mitochondrial DNA copy number of peripheral blood in bipolar disorder: the present study and a meta-analysis, Psychiatry Research (2018), doi: https://doi.org/10.1016/j.psychres.2018.08.014

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Highlights

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Bipolar disorder (BD) had low mitochondrial DNA copy number (mtDNAcn) than controls.

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Meta-analysis for all studies showed no association of mtDNAcn alteration with BD.

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Asian-specific meta-analysis showed lower mtDNAcn of BD than controls.

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Mitochondrial DNA copy number of peripheral blood in bipolar disorder: the present study and a

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meta-analysis

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Naruhisa Yamakia, Ikuo Otsukaa, Shusuke Numatab, Masaya Yanagic, Kentaro Mouria, Satoshi Okazakia,

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Shuken Bokua, Tadasu Horaia, Tetsuro Ohmorib, Osamu Shirakawac, Ichiro Soraa, Akitoyo Hishimotoa,*

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Biosciences, The University of Tokushima Graduate School, Tokushima, Japan

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Department of Psychiatry, Course of Integrated Brain Sciences, Medical Informatics, Institute of Health

Department of Neuropsychiatry, Kindai University Faculty of Medicine, Osaka, Japan

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Department of Psychiatry, Kobe University Graduate School of Medicine, Kobe, Japan

*Corresponding author: Akitoyo Hishimoto, M.D., Ph.D.

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Department of Psychiatry, Kobe University Graduate School of Medicine,

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7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

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TEL: +81-78-382-6065, FAX: +81-78-382-6079, E-mail: [email protected]

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Abstract

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Numerous evidence indicated mitochondrial abnormalities in the pathophysiology of bipolar disorder

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(BD); however, it remains unclear whether aberrant mitochondrial DNA (mtDNA) copy number (cn)

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occur in BD due to the conflicting results in previous studies. Here, peripheral blood mtDNAcn in 69 BD

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patients and 54 controls were analysed via qPCR. BD patients had significantly lower mtDNAcn

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compared to controls (regardless of their BD type [BD I or II]). Meta-analysis for all previous

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BD-mtDNAcn studies combining our results with previously published studies failed to identify any

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significant association. Meanwhile, Asian-specific meta-analysis remarkably revealed lower mtDNAcn

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in BD patients.

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(97 words)

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Keywords

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mitochondria, bipolar disorder (BD), meta-analysis

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1. Introduction

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Previous studies have implicated mitochondrial dysfunction in the pathogenesis of bipolar disorder

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(BD), by reporting decreased mitochondrial respiration, changes in mitochondrial morphology, increased

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incidences of mitochondrial DNA (mtDNA) polymorphisms and/or mutations, downregulated expression

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of molecules and proteins required for mitochondrial respiration, and abnormal high-energy phosphates

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(Morris et al., 2017; Young, 2007). mtDNA copy number (cn) alterations are considered to be indicative

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of mitochondrial dysfunction; however, only few studies have examined whether mtDNAcn changes

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occur in BD patients, and produced conflicting results (Chang et al., 2014; de Sousa et al., 2014; Fries et

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al., 2017; Wang et al., 2018).

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Here, first, peripheral blood mtDNAcn in 69 BD patients and age-matched 54 controls were analysed

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via qPCR. Further, a meta-analysis of the current and previous BD-mtDNAcn studies was performed, in

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order to mitigate the issue of small sample sizes within individual studies.

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2. Methods

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All participants were age-matched Japanese adults, comprising 69 BD patients and 54 healthy

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2005-2016. Sample characteristics are shown in Supplementary Table 1. All patients were currently

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diagnosed by two psychiatrists according to DSM-IV or DSM-5 criteria. Patients were excluded if they

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had 1) history of head trauma with a loss of consciousness, 2) current or previous neurological and/or

controls, that were recruited from the Kobe, Osaka, and Tokushima city areas of Japan between

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endocrine diseases, 4) alcohol/substance abuse or addiction history, 5) history of electroconvulsive

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therapy, 6) comorbid anxiety disorders. Healthy volunteers had no present, past, or family history of

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psychiatric disorders, or any current serious medical disorder. Written informed consent was obtained

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from all subjects for their participation in the present study, approved by the Ethics Committees for each

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institute. All procedures were performed in accordance with Declaration of Helsinki.

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mtDNAcn was calculated by using a quantitative polymerase chain reaction (qPCR) to measure and

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compare the level of mtDNA in each sample (by detecting NADH dehydrogenase, subunit 1; ND1) to

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that of a nuclear gene, β-hemoglobin (HGB). DNA extraction and qPCR experiment procedures were

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shown in Supplementary materials. mtDNAcn in each sample was determined by dividing cellular

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mtDNA content by cellular HGB content, and normalizing this ratio to that calculated for a reference

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sample using standard curve method with a 5-point serial-dilution series. The experiments were

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performed „blind‟, and the sample orders were randomized. Studies were selected for meta-analysis if they (1) measured mtDNAcn, (2) compared case (BD) with

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control subjects, (3) provided statistical analyses of generated mtDNAcn data, and (4) were published in

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English (The scheme and details of the study selection following the PRISMA guidelines are provided in

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Supplementary materials and Supplementary Fig 1).

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Statistical analyses were performed using R Version 3.2.2 and RevMan 5.3. Mann-Whitney test and

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Fisher‟s exact test were used to compare age and sex between groups, and regression analyses using

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generalized linear models were applied to determine whether mtDNAcn variations between groups with 5

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covariates (e.g. age and sex). A random effects model was used during the meta-analysis, due to the high

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heterogeneity (I2 = 81%, p < 0.01) between selected studies. Statistical significance was defined by

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two-tailed p < 0.05.

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3. Results

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There were no differences in the age distributions between groups (Supplementary Table 1).

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The present study showed significantly lower mtDNAcn in BD compared to controls (β = -0.138, p =

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0.025), and BD types (BD I or II) had no effect on mtDNAcn (Supplementary Table 2). Next, a meta-analysis was performed to combine this study with four previously published studies

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(Chang et al., 2014; de Sousa et al., 2014; Fries et al., 2017; Wang et al., 2018), producing overall 284

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BD patients and 201 control subjects; however, no significant differences in mtDNAcn between BD

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patients and controls were found (p = 0.64) (Fig. 1a). Meanwhile, in studies involving Asian cohorts, the

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Asian-specific meta-analysis revealed significantly lower mtDNAcn in BD patients than in controls (p <

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0.001) (Fig. 1b).

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4. Discussion

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The present study is the first to investigate mtDNAcn of BD patients compared to healthy volunteers

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in the Japanese population as well as to conduct meta-analyses involving all ethnicities or Asian-specific

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cohorts. Our results suggest reduced mtDNAcn in BD (regardless of their BD type [BD I or II]). Similar 6

to ours, some previous studies have reported lower mtDNAcn in peripheral blood of BD patients (Chang

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et al., 2014; Wang et al., 2018). Chang et al. (2014) reported that mtDNAcn was significantly lower in

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the BD euthymic patients. Wang et al. (2018) analysed the largest independent sample studied to date in

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BD-mtDNAcn field, and reported that both manic and depressed (but not the euthymic) states of BD are

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associated with lower mtDNAcn. On the other hand, de Sousa et al. (2014) and Fries et al. (2017)

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showed BD patients had no significant difference, and increased mtDNAcn compared to controls,

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respectively. Unsurprisingly, our meta-analysis involving the above previous studies above identified no

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significant differences between mtDNAcn exhibited by BD patients and controls. Such inconsistencies in

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BD-mtDNAcn studies could be attributed to the diversities in clinical features, tissue types and ethnicity.

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In fact, mitochondrial respiration and ATP production have been reported to be increased and decreased

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when patients experience bipolar-manic and euthymic/depressive state, respectively (Bai et al., 2015;

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Morris et al., 2017). Meanwhile, another study showed a marked decrease in complex II activity in

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peripheral blood mononuclear cells isolated from patients experiencing a depressed BD state compared

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to a euthymic BD state (Valvassori et al., 2018). Notably, the five studies analyzed by the current

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meta-analysis included a range of euthymic (Chang et al., 2014), depressed (de Sousa et al., 2014),

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Thus, further investigation is needed in each mood state. Mixed blood-cell populations should be

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considered as other limitation of this study and our meta-analysis (Supplementary Table 3), since the

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collection of whole-blood (rather than leukocyte) samples may have introduced variations in mtDNAcn

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three-classified (Wang et al., 2018) and non-classified patients (Fries et al., 2017 and the present study).

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via platelet contamination (Picard and McEwen, 2018). Interestingly, our Asian-specific meta-analysis

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for BD-mtDNAcn studies revealed significant lower mtDNAcn in BD patients with a low level of

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heterogeneity (I2 = 29%, p > 0.05). Thus, future BD-mtDNA studies need to subdivide the study

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population into different ethnic populations because the construction and function of mtDNA has

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remarkable diversity in each ethnicity (known as “mtDNA haplogroup”) (Santoro et al., 2006). In

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addition, our results may be confounded by undisclosed lifestyle factors known to affect mtDNAcn, such

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as smoking status, psychiatric rating scales, number of episodes and BMI (Révész et al., 2018).

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Furthermore, the biological importance of mtDNAcn increases/decreases in peripheral blood and whether

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any correlation exists between blood mtDNAcn and brain mtDNAcn are often unclear, and therefore

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difficult to interpret. Future analyses of mtDNAcn in the brain tissues of BD may be useful to more

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directly investigate the relationship between the brain mtDNAcn and BD pathogenesis.

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This work was supported in part by funding from JSPS KAKENHI (17H04249).

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Acknowledgements

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Conflict of interest All authors declare no conflict of interest.

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Funding Sources 8

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This work was partly supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 17H04249).

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Figure Legend Fig 1. Forest plots depicting the results of the conducted meta-analysis investigating the effects of

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mitochondrial DNA copy number (mtDNAcn) variations in BD patients and control subjects. a) The meta-analysis combining this study with four previously published studies (Chang et al., 2014; de Sousa et al., 2014; Fries et al., 2017; Wang et al., 2018).

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b) The meta-analysis combining this study only with Asian cohort studies (Chang et al., 2014; Wang et al., 2018).

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BD, bipolar disorder; CON, control; CI, confidence interval; SD, standard deviation.

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Fig. 1.

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