Sequence and functional analyses of mtDNA in a maternally inherited family with bipolar disorder and depression

Mutation Research 617 (2007) 119–124

Sequence and functional analyses of mtDNA in a maternally inherited family with bipolar disorder and depression Kae Munakata a , Kumiko Fujii b , Shinichiro Nanko c , Hiroshi Kunugi d , Tadafumi Kato a,∗ a

c

Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan b Department of Psychiatry, Shiga University of Medical Science, Otsu 520-2192, Japan Department of Psychiatry, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi, Tokyo 173-8605, Japan d Department of Mental Disorder Research, National Institute of Neuroscience, 4-1-1, Ogawahigashi, Kodaira 187-8502, Japan Received 26 May 2006; received in revised form 4 January 2007; accepted 17 January 2007 Available online 21 January 2007

Abstract Recent studies suggest that mutations/polymorphisms of mitochondrial DNA (mtDNA) are associated with neuropsychiatric diseases. We identified a patient with major depression and epilepsy. Some family members in the pedigree of the proband had bipolar disorder, depression, suicide, or psychotic disorder not otherwise specified. The mode of inheritance was compatible with maternal inheritance with low penetration. We assumed that the mental disorder in this family might be associated with maternally inherited mitochondrial DNA (mtDNA) mutation. We sequenced the entire mtDNA of the proband. Among the 34 base substitutions detected in the proband, two homoplasmic, nonsynonymous single substitutions of mtDNA, T3394C in MT-ND1 and A9115G in MT-ATP6, were suspected to cause functional impairment, because the former was reported to be disease-related and the latter is vary rare. To study the functional outcome of these substitutions, we examined mitochondrial membrane potential and the activity of mitochondrial ATP synthesis in the transmitochondrial cybrids, but no significant impairment was detected. The data did not support our hypothesis that these disorders in this family are caused by mtDNA mutation(s). © 2007 Elsevier B.V. All rights reserved. Keywords: mtDNA; Bipolar disorder; Depression

1. Introduction Bipolar disorder is a major mental disorder characterized by recurrent manic and depressive episodes. From twin, family and linkage studies, the role of mulAbbreviations: OXPHOS, oxidative phosphorylation Corresponding author. Tel.: +81 48 467 6949; fax: +81 48 467 6947. E-mail address: [email protected] (T. Kato). ∗

0027-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2007.01.006

tifactorial genetic factors is well established; however, in spite of extensive studies, no causative genes have been established. Three lines of evidence suggest the possible involvement of mitochondrial DNA in bipolar disorder: (1) mitochondrial dysfunction in the brains of patients with bipolar disorder detected by magnetic resonance spectroscopy (MRS) [1], (2) the possible involvement of maternal inheritance in bipolar disorder [2], and (3) comorbidity of mood disorder in patients with mtDNA

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mutations [3–5]. More recently, the predisposition to depression in mothers or other matrilineal relatives of patients with maternally inherited mitochondrial diseases was reported [6]. Based on these findings, we proposed the mitochondrial dysfunction hypothesis of bipolar disorder [7], and have been studying the possible roles of mitochondrial DNA in bipolar disorder [8]. Recent other studies supported our hypothesis: increased lactate in the brain detected by MRS [9], altered expression of mitochondria-related genes in postmortem brains [10], and comorbidity of mood disorders with mitochondrial diseases [11]. We have found several mutations/polymorphisms of mitochondrial DNA (mtDNA) associated with bipolar disorder and other mental disorders [12–16]. Some of these mtDNA polymorphisms/mutations were suggested to cause functional impairment [13,17–19]. During the course of our study, we identified a proband suffering from major depression and epilepsy. Some family members of this patient had mental disorders such as bipolar disorder. The mode of inheritance of mental disorder in this family was compatible with maternal inheritance with low penetration. We speculated that mitochondrial DNA mutations might have caused mental disorders in this family. Here, we sequenced the entire mtDNA of the proband and identified two putative disease-related base substitutions of mtDNA (T3394C and A9115G). However, a case–control study and functional analyses using transmitochondrial hybrid cells (cybrids) did not show a robust association of these mutations with bipolar disorder. Several possibilities accounting for these negative findings are discussed.

2. Subjects and methods 2.1. Profile of the family The proband was 48 years old on participation in this study (Fig. 1, II-5). At the age of 47, after severe psychological stress, she became confused and visited a psychiatrist. She was diagnosed with a “psychotic disorder not otherwise specified.” Four months later, she showed major depression and was treated with anxiolytics for about 1 month. After initial relief, her depression relapsed with psychotic features 8 months later. She was admitted to a psychiatric ward at the age of 51. During hospitalization, she had epilepsy with various forms of seizures during antidepressant treatment. Typical seizures of this patient began with the rotation of the face to the left side, followed by left side gaze, clonic seizure of left arm, and end with the face to the right side. However, the forms of the seizures were not consistent. A similar episode occurred 3 months later after the antidepressants had been stopped. It was unlikely that she took unexpected medicine for suicide, even though we did not perform drug test, because the patient was in the hospital at that time, and under the nurse-care management. Electroencephalogram showed polyspikes in the frontal region. 123 I-isopropyl iodoamphetamine (IMP)-SPECT (single photon emission tomography) showed reduction of blood flow in the bilateral frontal cortex. She gradually developed frontal lobe syndrome. Muscle biopsy revealed neurogenic muscle atrophy but did not show ragged-red fibers by Gomori-Trichrome staining; therefore, MERRF (mitochondrial myopathy, with ragged-red fibers) was ruled out. All six siblings of the proband were interviewed by an experienced psychiatrist and diagnosis was made according to DSM-III-R criteria. Two had bipolar disorder, and one had recurrent major depression. The mother (I-3) suffered bipolar disorder and her sister (I-4) committed suicide, suggesting depression. Both a daughter and a son of the sister (II-8, II-9) showed psychotic disorder not otherwise specified. DNA samples of the proband and her siblings were

Fig. 1. The pedigree of the family. We sequenced the entire 16.6 kb mtDNA of the proband with major depression. Abbreviations: BP, bipolar disorder; MD, major depression; P-NOS, psychotic disorder not otherwise specified. In addition, the following members suffered physical symptoms—II-1, Migraine with aura, gastric resection due to gastric ulcer; II-3, headache; II-6, headache, gastric ulcer. In generation I, each brother in one family married each sister in another family. The number in rhomboid shows the number of siblings of unknown gender.

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obtained. The following molecular studies were approved by the ethics committees of RIKEN and Shiga University. 2.2. Entire mtDNA sequencing Total DNA was isolated from leukocytes of the proband and the entire mtDNA was sequenced as previously described [13]. The sequence was verified at least twice in both directions, and was compared with the revised Cambridge reference sequence (rCRS, accession no. GI: 337188). Base substitutions were determined as to how they affect the resulting amino acids using MitoAnalyzer (http://www.cstl.nist.gov/biotech/strbase/mitoanalyzer.html). 2.3. Case–control study The sample set used for the case–control study consists of 199 patients with bipolar disorder (143 bipolar I disorder and 56 bipolar II, 76 males and 123 females, 49.8 years old on average) and 255 control subjects (129 males and 129 females, 33.0 years of age on average). Patients with bipolar disorder were diagnosed according to the DSM-III-R or DSM-IV criteria by at least two interview sessions by two senior psychiatrists, and a consensus diagnosis was made. Their family history of mental disorder was assessed by interviewing the proband and available relatives. Control subjects were recruited from the staff or students of participating institutes and their friends, who reported themselves to be healthy. Written informed consent was obtained from all subjects. The detected mtDNA mutations were genotyped by PCRRFLP. To detect 9115A/G; we used forward mismatch primer, 5 -CACTTATCATCTTCACAATTCGA-3 ; reverse primer, 5 -GTCATGGGCTGGGTTTTACT-3 , and restriction enzyme EcoRI. To detect 3394T/C, forward primer, 5 -AGGACAAGAGAAATAAGGCC-3 ; reverse primer, 5 AGAAGAGCGATGGTGAGAGC-3 , and restriction enzyme, HaeIII, were used. 2.4. Cell culture To examine the functional consequence of mitochondrial DNA mutations, we obtained cybrids containing the mtDNA of the proband using 143B·TK− ␳0 cells lacking mtDNA and platelets. We also used four cybrid cell lines, previously generated as controls [13]. Cybrids were cultured with DMEM containing 10% fetal bovine serum, penicillin/streptomycin (all from GIBCO BRL). As a functionally negative control, we cultured 143B·TK− ␳0 cells with DMEM containing 10% fetal bovine serum, penicillin/streptomycin, and 50 ␮g/ml uridine as previously described [20]. 2.5. Evaluation of mitochondrial functions in cybrids To confirm the integration of mtDNA in the fused cybrid, we performed Southern blotting as previously described [13]. Next, the mtSNPs were checked as described above. Con-

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trol cybrids were selected from samples generated previously, so as to match the Japanese sub-haplogroup, 5178C/10398G [21], to the cybrids of the proband. To examine the integration of each complex of OXPHOS and their enzyme activity, especially complexes I and V, we analyzed the protein complexes of OXPHOS using blue-native PAGE. The whole procedure of blue-native PAGE and in gel activity was performed as previously described [22]. Evaluation of the mitochondrial membrane potential of cybrids was performed using JC-1 as previously described [13]. The activity of the ATP synthetase of cybrids was measured by luminometric determination, previously described by Manfredi et al. [23], with some modification. In short, we used ENLITEN (Promega), previously mixed and optimized with luciferin and luciferase. In the assay of ATP production, 170 ␮l of the suspension of 1 × 105 of digitonin-permeabilized cells, 5 ␮l of 6 mM DAPP (P1,P5-di(adenosine-5 ) pentaphosphate pentasodium salt, CAS Number: 4097-04-5), 5 ␮l of a substrate mixture of either 80 mM pyruvate (GIBCO BRL) and 80 mM malate, or 400 mM succinate and 500 ␮g/ml rotenone, with or without 2 ␮g/ml oligomycine, and 20 ␮l of reconstituted ENLITEN: ATP production was measured by the increase of relative light units (RLU) after adding the 0.1 mM ADP, using a luminometer, GENE LIGHT 55 (Microtec Co. Ltd., Chiba, Japan). Digitonin, DAPP, malate, succinate, rotenone and oligomycine were obtained from Sigma–Aldrich, Japan.

3. Results 3.1. MtDNA substitutions of T3394C and A9115G Before sequencing mtDNA, we performed longPCR to check if there was any large-scale deletion of mtDNA in the sample [24], and no deletion was detected. Comparing the human standard sequence of mtDNA (accession no. GI: 337188), 34 base substitutions were identified in the sequence of the proband: 11 of D-loop region, three of rRNA, 13 of synonymous substitutions, and seven of nonsynonymous substitutions (Table 1). Thirty-two had been registered as polymorphisms in the mtDNA database MITOMAP (http://www.mitomap.org/) [25]. One of the nonsynonymous base substitutions found in the proband, T3394C, converting tyrosine to histidine in MT-ND1 of complex I (Grantham value by the change, 83), was reported as a disease-associated mutation related to non-insulin dependent diabetes mellitus (NIDDM) and Leber’s hereditary optic neuropathy (LHON). The other nonsynonymous substitution, A9115G, converting isoleucine to valine in MT-ATP6 of complex V (Grantham value by the change, 29), had not been registered in MITOMAP. In the Japanese mtDNA database, GiiB-JST mtSNP (http://www.giib.or.jp/mtsnp/index e.shtml) [26], both have been found in several Japanese. While T3394C was

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Table 1 Evolutionary conservation of the changed amino acids (a) Protein sequence of ND1 Patient (T3394C, Y30H) Human Gorilla Bovine Mouse Urchin Drosophilae

FLMLTERKILGHMQLRKGPNVVGPY FLMLTERKILGYMQLRKGPNVVGPY FLMLTERKILGYMQLRKGPNVVGPY FLTLVERKVLGYMQLRKGPNVVGPY FLTLVERKILGYMQLRKGPNIVGPY FLTLVERKVLGYMQFRKGPKVVGPY FLTLLERKVLGYIQIRKGPNKVGLM

(b) Protein sequence of ATP6 Patient (A9115G, I197V) Human Gorilla Bovine Mouse Urchin Drosophilae

PSTLIIFTILVLLT-I-LEIAVAL PSTLIIFTILILLT-I-LEIAVAL PSTLIIFTVLILLT-M-LEIAVAL TTALITFTILILLT-I-LEFAVAM PTATITFIILLLLT-I-LEFAVAL SIPLFL–IFVLLF-I-LEIGVAC FL-LVAQ-I-ALLV–LESAVTM

Table 2 Case–control study Genotype

found in one centenarian, A9115G was not found in 96 centenarians and 198 young healthy subjects. 3.2. Putative consequence of the substitutions and evolutional conservation The base substitution, T3394C, converts the 30th amino acid, tyrosine, to histidine in the protein subunit MT-ND1. In phylogenic comparison, the 30th tyrosine is well conserved from Drosophila to humans (Table 1), including 61 mammalian species (http://www.giib.or.jp/ mtsnp/search mtSAP evaluation e.html). Although the role of MT-ND1 in complex I is not well known, we previously reported that T3644C in MT-ND1 modestly impaired the activity of complex I [13]; therefore, we suspected that this base substitution might cause functional impairment. A9115G is located in MT-ATP6 and converts 197th isoleucine to valine. The 197th isoleucine is not well conserved in 61 mammalian species (http://www.giib.or.jp/ mtsnp/search mtSAP evaluation e.html). We further examined the functional consequence of these mutations using cybrids. 3.3. Allele frequencies of T3394C and A9115G in Japanese patients with bipolar disorder The results of association studies are described in Table 2. Regarding T3394C, there was no significant difference in the frequency of 3394C between bipolar patients and controls by Fisher’s exact test. In MITOMAP (http://www.mitomap.org), 3394C was associated with NIDDM and LHON; however, in the Japanese mtSNP database (http://www.giib.or.jp/mtsnp/

3394T 3394C Bipolar disorder Control

192 250

7 5

9115A 9115G 198 p = 0.38 255

1 0

p = 0.44

p value is calculated by Fisher’s exact test.

index e.shtml), 4/96 people with NIDDM, 4/96 thin young males, 3/96 obese young males, 2/96 with Parkinson disease, and 1/96 centenarians had 3394C; thus, the pathological significance of this base substitution is unlikely. The patient with 9115G in the case–control study of bipolar disorder was the brother of the proband. According to the Japanese mtSNP database (http://www.giib.or.jp/mtsnp/index e.shtml), only one Japanese with NIDDM/angiopathy (1/96) and one with Parkinson disease (1/96) had 9115G. Although the result of the case–control study indicated no significant association of these mtSNPs with bipolar disorder, it is difficult to rule out the possible pathophysiological significance of this rare mutation. 3.4. Analyses of OXPHOS in transmitochondrial cybrids containing mtDNA of the patient To minimize the effect of large inter-individual variation of mtDNA, we used four control cybrids containing the mtDNA sequences with a similar macro-haplogroup, 5178C/10398G, as described above. We confirmed that these cybrids had the same genotype as the donor subjects, and that all of these cybrids maintained the mtDNA at a comparable level by Southern blotting (data not shown). The migration image of complexes of OXPHOS in blue-native PAGE was similar to that of controls (data not shown). The results comparing the mitochondrial membrane potential (MMP) between the cybrids from the proband and controls are described in Table 3. Although statistical analysis cannot be applied, the cybrid derived from the proband showed a similar function to controls. Regarding the ATP synthesis of these cybrids, we measured two types of ATP synthesis: Table 3 Mitochondrial membrane potential of cybrids Genotype (number)

Energized mitochondria (%)

3394T/9115A (7) 3394C/9115G (1a ) ␳0 cells (1)

80.7 ± 10.3 73.6 13.2

a

The cybrid cell line obtained from the proband.

K. Munakata et al. / Mutation Research 617 (2007) 119–124 Table 4 ATP synthetase activity Genotype (number)

3394T/9115A (4) 3394C/9115G (1a ) ␳0 cells (1) a

Total cell protein (pmol/[min mg]) Pyruvate + malate

Succinate + rotenone

134.0 ± 47.6 99.1 5.7

104.4 ± 44.5 106.0 3.6

The cybrid cell line obtained from the proband.

one is driven and coupled from complex I, pyruvate and malate, and the other from complex II, rotenone and succinate. The activity of ATP synthetase in cybrids with 3394C/9115G was kept at a similar level to that in control cybrids, either by using pyruvate and malate as substrates, or using succinate as a substrate with the presence of complex I inhibitor, rotenone (Table 4). These results indicated that MMP was maintained and ATP production was almost intact in cybrids with 3394C/9115G. 4. Discussion We speculated that mtDNA mutation(s) may be causative for mental disorder in this family because: (1) the proband with major depression also suffered from various forms of seizures, which can be seen in a typical mitochondrial disorder, MERRF (myoclonic epilepsy and ragged-red muscle fibers) (NIM 545000); (2) the mode of inheritance was compatible with maternal inheritance with low penetration. In addition, one of the sisters with major depression suffered migraine with aura, which is reportedly associated with mtDNA mutations [27,28]. It was unlikely that the possibility of toxicity or abrupt cessation of antidepressant that is related with the episodes of seizure because all the epileptic seizures occurred in the hospital and under the strict nurse–case management. By sequencing the whole mtDNA of the proband, we identified two candidate base substitutions. The 3394C altering evolutionally conserved amino acid in complex I was previously reported to be associated with diabetes mellitus. The 9115G mutation causing amino acid substitution in complex V was not found in any healthy controls but was reported in two patients, Alzheimer’s disease or diabetes mellitus, in the mtSNP database. A case–control association study, however, did not show a robust association of these mtDNA substitutions with bipolar disorder. As the results of the association study were not conclusive due to the low frequency of these mutations, we further tested the possible significance of these two base substitutions. We generated transmitochondrial cybrids containing mtDNA of the proband and measured MMP

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and complex V activity; however, no marked alteration in MMP or complex V activity was found. Several interpretations can be made for these findings: (1) The disorders in this family were not caused by maternal inheritance but by autosomal dominant or multifactorial inheritance. This is the most likely interpretation because both the association study and functional analysis suggest that these base substitutions have no pathophysiological significance. (2) The symptoms of these patients were caused by heteroplasmic mtDNA mutations, which were overlooked in this study. This is possible because we used DNA only from leukocyte DNA, but could not use DNA extracted from biopsied muscles. (3) Subtle functional alteration caused by these two substitutions could not be detected in the nuclear background of 143B·TK− . This possibility cannot be ruled out because it has been suggested that nuclear background can affect the expression of phenotypes associated with mtDNA polymorphisms [29,30]. (4) These two substitutions may cause functional alteration in other mitochondrial functions, such as calcium signaling, but do not cause impairment in the electron transport chain. Although the possible significance of these two mtDNA base substitutions cannot be totally ruled out as discussed above, these negative results caution us against speculating functional impairment caused by unknown nonsynonymous mtDNA substitutions without functional analysis. References [1] T. Kato, S. Takahashi, T. Shioiri, T. Inubushi, Alterations in brain phosphorous metabolism in bipolar disorder detected by in vivo 31P and 7Li magnetic resonance spectroscopy, J. Affect. Disord. 27 (1993) 53–59. [2] F.J. McMahon, O.C. Stine, D.A. Meyers, S.G. Simpson, J.R. DePaulo, Patterns of maternal transmission in bipolar affective disorder, Am. J. Hum. Genet. 56 (1995) 1277–1286. [3] G. Siciliano, A. Tessa, S. Petrini, M. Mancuso, C. Bruno, G.S. Grieco, A. Malandrini, L. DeFlorio, B. Martini, A. Federico, et al., Autosomal dominant external ophthalmoplegia and bipolar affective disorder associated with a mutation in the ANT1 gene, Neuromuscul. Disord. 13 (2003) 162–165. [4] J.N. Spelbrink, F.Y. Li, V. Tiranti, K. Nikali, Q.P. Yuan, M. Tariq, S. Wanrooij, N. Garrido, G. Comi, L. Morandi, et al., Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria, Nat. Genet. 28 (2001) 223–231. [5] G. Van Goethem, B. Dermaut, A. Lofgren, J.J. Martin, C. Van Broeckhoven, Mutation of POLG is associated with progres-

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