Analysis of mitochondrial DNA variants in Japanese patients with schizophrenia

Mitochondrion 9 (2009) 385–393

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Analysis of mitochondrial DNA variants in Japanese patients with schizophrenia Hitomi Ueno a,b, Yutaka Nishigaki a,*, Qing-Peng Kong c, Noriyuki Fuku a, Shuji Kojima b, Nakao Iwata d, Norio Ozaki e, Masashi Tanaka a a

Department of Genomics for Longevity and Health, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan c State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China d Department of Psychiatry, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake-shi, Aichi 470-1192, Japan e Department of Psychiatry, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa-Ku, Nagoya-shi, Aichi 466-8550, Japan b

a r t i c l e

i n f o

Article history: Received 30 January 2009 Received in revised form 8 May 2009 Accepted 22 June 2009 Available online 27 June 2009 Keywords: Mitochondrial DNA Schizophrenia Polymorphism Variant Mutation

a b s t r a c t To test the hypothesis that mitochondrial DNA (mtDNA) variants contribute to the susceptibility to schizophrenia, we sequenced the entire mtDNAs from 93 Japanese schizophrenic patients. Three nonsynonymous homoplasmic variants in subunit six of the ATP synthase (MT-ATP6) gene that were detected only in patients but not in controls were suggested to be slightly deleterious, because (1) their original amino acid residues (AA) were highly conserved and (2) the physicochemical differences between the original and altered AA were relatively high. In addition, we detected three novel heteroplasmic variants that were potentially pathogenic. Although functional analysis is needed, rare variants in the mtDNA may convey susceptibility to schizophrenia. Ó 2009 Elsevier B.V. on behalf of Mitochondria Research Society. All rights reserved.

1. Introduction The genetic transmission of schizophrenia does not appear to follow simple Mendelian single-gene inheritance patterns. It has been suggested that there are multiple susceptibility loci, each having a small effect and acting in concert with epigenetic and environmental factors (Mueser and McGurk, 2004). Predicting the genetic risk for schizophrenia is very important, because it may lead to the early detection and proactive management of this condition. Enormous efforts have gone into elucidating the contribution of genetics to schizophrenia, mainly based on the hypothesis that common diseases are caused by various combinations of common variants in the population (Allen et al., 2008; O’Donovan et al., 2008). In contrast, recent studies focusing on rare variants demonstrated that individually rare structural variants or copy number variations (CNVs) contributed to the risk of schizophrenia (Walsh et al., 2008; Xu et al., 2008). It is possible that a common phenotype such as schizophrenia can result from a variety of individually rare variants in the different genes and their interaction with various environmental factors. The genetic basis of schizophrenia seems to be very complex and preceding studies focusing on the nuclear genome have revealed only a part of the genetic basis of the disease. * Corresponding author. Tel.: +81 3 3964 3241x3096; fax: +81 3 3579 4776. E-mail address: [email protected] (Y. Nishigaki).

In the present study, we focused on the mitochondrial DNA (mtDNA) to identify the genes associated with the development of schizophrenia. This study aimed at elucidating the genetic basis underlying the altered gene expression (Karry et al., 2004), as well as the structural (Kung and Roberts, 1999) and biochemical (Cavelier et al., 1995) abnormalities found in mitochondria of schizophrenic patients. Prabakaran et al. (2004) proposed that oxidative stress and the ensuing cellular adaptations could be linked to the schizophrenia disease process, based on their study on prefrontal cortex tissue obtained from autopsied schizophrenia individuals. Doi and Hoshi (2007) argued that the model of multiple genes in nuclear DNA cannot account for the persistence problem in schizophrenia without unrealistic assumptions. Instead, they hypothesized that de novo mutations in the mitochondrial genome could account for this persistence problem. Furthermore, schizophrenia and other psychotic disorders share several features with mitochondrial diseases arising as a result of mtDNA mutations (Prayson and Wang, 1998). These lines of evidence implicate mtDNA as having a role in the pathogenesis of schizophrenia. We recently described the association of major mitochondrial haplogroups with metabolic syndrome (Tanaka et al., 2007), type 2 diabetes mellitus (Fuku et al., 2007), and atherosclerosis-related diseases such as myocardial infarction (Nishigaki et al., 2007b) and atherothrombotic cerebral infarction (Nishigaki et al., 2007a) on the basis of the comprehensive analysis of mitochondrial single nucleotide polymorphisms (mtSNPs) in the coding region of mtDNA. Thus,

1567-7249/$ - see front matter Ó 2009 Elsevier B.V. on behalf of Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2009.06.003

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H. Ueno et al. / Mitochondrion 9 (2009) 385–393

we hypothesized that common or rare variants of mtDNA are associated with the schizophrenic pathogenesis. To test this hypothesis, we initially determined the entire sequences of mtDNA from 93 schizophrenic patients. Then, we compared the frequencies of mitochondrial major haplogroups between schizophrenic patients and 784 Japanese control subjects. Subsequently, we evaluated the effects of rare homoplasmic or heteroplasmic substitutions detected only in schizophrenic patients but not in Japanese controls by estimating their effects on the secondary structures of gene products and their conservation among different species.

tify possible mtSNP loci and mutations, and to compare the sequences with the revised Cambridge reference sequences (Anderson et al., 1981; Andrews et al., 1999). To avoid potential problems, we followed the previously proposed suggestions (Kong et al., 2008) during the data generation. Based on the obtained variations, all the individuals were allocated to the smallest clades on the reconstructed East Asian mtDNA tree (Kong et al., 2006; Tanaka et al., 2004). Newly obtained mtDNA sequences of schizophrenic patients were deposited in GenBank as the following accession numbers: AP010970–AP011059.

2. Materials and methods

2.3. Analysis by PCR-restriction fragment length polymorphism (RFLP) method

2.1. Patients and control subjects The study population comprised 93 Japanese patients with schizophrenia who either visited outpatient clinics of or were admitted to one of the participating hospitals. Schizophrenia was defined according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). Details about the background (sex, age, and family history) of the patients are given in Table 1. Among these subjects, each of the following three pairs belongs to the same maternal family: SPsq0010-SPsq0058, SPsq0067SPsq0068, and SPsq0072-SPsq0073. The study protocol was approved by the Ethical Committees of Tokyo Metropolitan Institute of Gerontology and Fujita Health University School of Medicine, and informed consent was obtained from each participant. The non-schizophrenic control group comprised 784 unrelated Japanese individuals. The entire mitochondrial genomes of these subjects were previously determined by us (Alexe et al., 2007; Bilal et al., 2008; Tanaka et al., 2004), and the data can be found at the mitochondrial SNP database (http://mtsnp.tmig.or.jp/mtsnp/ index_e.shtml). 2.2. Sequence analysis of mtDNA Genomic DNA was extracted from the peripheral blood of 93 schizophrenic patients. The entire mitochondrial genome was amplified as six fragments (approximately 3 kb) by the first polymerase chain reaction (PCR) and 60 overlapping segments (approximately 600 bp) by the second PCR. The primer pairs and PCR conditions were described previously (Tanaka et al., 2002). These second PCR products were purified with MultiScreen-PCR Plates (Millipore, Billerica, MA). Sequence reactions were carried out with a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). Sequences were analyzed with an Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems) and Sequencher version 4.2.2. (Gene Codes Co, Ann Arbor, MI) to iden-

Table 1 Background of the 93 patients with schizophrenia. 39a 54

Sex

Male Female

Age (yrs.)

Under 20 20’s 30’s 40’s Over 50 Unknown

2 15 21 7 3 45

Family history

+ – Unknown

18b 29 46

a

Number of the schizophrenic patients. Three pairs (SPsq0010-SPsq0058, SPsq0067-SPsq0068, SPsq0073) were maternally related. b

and

SPsq0072-

To confirm the heteroplasmic variants m.1227G > A in the 12S ribosomal RNA (MT-RNR1) gene and m.13418G > A (Gly361Glu) in the NADH:ubiquinone oxidoreductase subunit 5 (MT-ND5) gene, we performed restriction fragment length polymorphism (RFLP) analysis. For detection of the m.1227G > A variant, a 135bp mtDNA fragment spanning nucleotides 1129–1263 was amplified from total DNA by using the forward primer 50 -TGCTCGCCAG AACACTACGAGCCACA-30 (nucleotides 1129–1154) and the reverse mismatch primer 50 -CGGTATATAGGCTGAGCAAGAGGTGGTGAGG TTAAT-30 (nucleotides 1228–1263, with mismatch nucleotide underlined). The m.1227G > A mutation introduces a restriction site of Tsp509I (New England BioLabs Inc., Beverly, MA) that is detectable by RFLP analysis. The fragment (135 bp) with the G–A mutation at nucleotide 1227 was cut with Tsp509I into two fragments (98 bp and 37 bp), whereas the wild-type mtDNA fragment was not restricted with the enzyme. Digested PCR products were electrophoresed in 4% NuSieve 3:1 agarose gels (data not shown). For the detection of the m.13418G > A variant, a 149-bp mtDNA fragment spanning nucleotides 13308–13456 was amplified from total DNA by using the forward primer 50 -AGCATTCCTGCACATCT GTACCCACG-30 (nucleotides 13308–13333) and the reverse mismatch primer 50 -TGAGGGAGGTTGAAGTGAGAGGTATGGTTTTGAGT AAT-30 (nucleotides 13419–13456, with mismatch nucleotide underlined). The m.13418G > A mutation introduces a Tsp509I restriction site that is detectable by RFLP analysis. The fragment (149 bp) with the G–A mutation at nucleotide 13418 was cut with Tsp509I into two fragments (110 bp and 39 bp), whereas the wildtype mtDNA fragment was not restricted with the enzyme. Digested PCR products were electrophoresed in 4% NuSieve 3:1 agarose gels (data not shown). 2.4. Quantification of the mutation loads for heteroplasmic variants To quantify the levels of the two heteroplasmic variants, m.5578T > C, in the transfer RNA tryptophan (MT-TW) gene and m.13418G > A in the MT-ND5 gene in the mtDNA, we cloned and sequenced the PCR-amplified DNA fragments flanking the mutation sites. For the analysis of m.5578T > C, a DNA fragment encompassing nucleotides 5545–6028 was amplified by PCR from total DNA by using the forward primer 50 -ACAGCTAAGGACTGCAAAAC-30 (nucleotides 5545–5564), the reverse primer 50 -CCCAGCTCGGCT CGAATAAG-30 (nucleotides 6009–6028), and Platinum Taq DNA polymerase high fidelity (Invitrogen, Carlsbad, CA). For the analysis of m.13418G > A, a DNA fragment encompassing nucleotides 13308–13786 was amplified by PCR from total DNA with the forward primer 50 -AGCATTCCTGCACATCTGTACCCACG-30 (nucleotides 13308–13333) and the reverse primer 50 -GGGGGATTGTTGTTTGG AAG-30 (nucleotides 13767–13786). The PCR cycle conditions were the following: 1 cycle at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min; and an extension cycle at 68 °C for 7 min. The PCR products were isolated after separation by electrophoresis in 2% agarose gels. Each of the PCR frag-

H. Ueno et al. / Mitochondrion 9 (2009) 385–393

ments was extracted from the agarose gel by using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), ligated into the pCR II-TOPO vector, and subcloned by using a TOPO TA Cloning Kit (Invitrogen). Approximately 50 cloned plasmids of each PCR product were purified with a High Pure Plasmid Isolation Kit (Roche Diagnostic Corp., Indianapolis, IN), and sequenced directly in an Applied Biosystems 3130xl Genetic Analyzer following the procedures described previously (Nishigaki et al., 2003a). 2.5. Two-dimensional structures of mitochondrial proteins, rRNA, and tRNA The two-dimensional structures of mitochondrial proteins are displayed in our web site (https://www.ilh.jp/mtsnp/search_ mtSAP_2D_e.html), with the structures predicted according to the SOSUI system (http://bp.nuap.nagoya-u.ac.jp/sosui/). The secondary structure of tRNA is displayed as the conventional cloverleaf structure (http://mtsnp.tmig.or.jp/cgi-bin/mtsnp/2DStruct/ tRNA2DStructure_fw.cgi). The core secondary structure model for mammalian 12S rRNA (MT-RNR1) was provided by Springer and Douzery (1996), who used the Bos Taurus sequence. 3. Results 3.1. Mitochondrial haplogroup distribution of 93 schizophrenic patients All mtDNA variations detected in 93 subjects and haplogroup classification are shown as the mtDNA phylogenetic tree in Fig. 1. Table 2 shows the distribution frequencies of the 10 Japanese major haplogroups: A, B, D4, D5, F, G1, G2, N9a, M7a, and M7b (Tanaka et al., 2007). We excluded three subjects (SPsq0058, SPsq0068, and SPsq0073) from subsequent analysis because three pairs (i.e., SPsq0010-SPsq0058, SPsq0067-SPsq0068, and SPsq0072SPsq0073) were maternally related and their whole mtDNA sequences were identical. Fisher’s exact test revealed that the frequencies of these haplogroups were not significantly different between 90 schizophrenic patients and 784 Japanese control subjects on the basis of a P value of <0.05 (Table 2). 3.2. Individually rare homoplasmic substitutions in mtDNA from schizophrenic patients Individually rare homoplasmic substitutions that were detected in 1 or 2 schizophrenic patients but not in the 784 controls are manifested on the terminal branches of the phylogenetic tree (Fig. 1). Among these variants, the functional effects of non-synonymous substitutions were estimated by determining their conservation among 61 different mammalian species. We found five rare homoplasmic variants: m.3472T > C (Phe56Leu) in the MT-ND1 gene, m.8843T > C (Ile106Thr), m.8902G > A (Ala126Thr) and m.8945T > C (Met140Thr) in the subunit six of the ATP synthase (MT-ATP6) gene, and m.9604A > G (Asn133Ser) in the cytochrome c oxidase subunit III (MT-CO3) gene, which were highly conserved (>90%) among 61 mammalian species (Table 3). The 8843T > C substitution (Ile106Thr) in the MT-ATP6 gene was detected in one schizophrenic patient (SPsq0080) but not in the 784 Japanese controls. This substitution was observed in four European subjects (FNsq9406, MKsq0267, MKsq0289, and MKsq0456 in the mtSNP database). SPsq0080 was classified into B4c1a1a, whereas the other four European subjects harboring m.8843T > C did not belong to haplogroup B4c1. It is therefore possible that m.8843T > C is an individually rare variant rather than a common polymorphism characteristic for a certain mitochondrial haplogroup. Ile106 is located in the transmembrane domain of the p.MT-ATP6 subunit (Fig. 2) and is highly conserved among 56

387

mammalian species (Table 3). Threonine, as a polar amino acid residue, was not found at amino acid position 106 among the 61 mammalian species. The m.8902G > A substitution (Ala126Thr) in the MT-ATP6 gene was reported in none of the three databases i.e., the mtSNP database, the MITOMAP (http://www.mitomap.org/), and the mtDB (http://www.genpat.uu.se/mtDB/) as of October 2008. Amino acid position 126 is located in the inter membrane side of the p.MTATP6 subunit (Fig. 2). The Ala126 was strictly conserved among 59 mammalian species (Table 3). Echinops telfairi (small Madagascar hedgehog) had a serine residue and Pongo pygmaeus abelii (Sumatran orangutan) had a threonine residue at this position. The m.8945T > C replacement (Met140Thr) in the MT-ATP6 gene was also reported in none of the three databases. As shown in Fig. 2, the amino acid position 140 is located in the transmembrane domain of p.MT-ATP6. Met140 is strictly conserved among 60 of 61 mammals (Table 3). Although a leucine residue was found at this position in Macaca sylvanus (Barbary ape), a threonine residue as a polar amino acid residue was not found among the examined mammalian species. Although the effects of these three amino acid replacements on the function of FOF1-ATPase are unknown, it is possible that these replacements might be slightly deleterious, because the amino acid replacements occurred at the highly conserved (>90%) sites as well as because their physicochemical differences between the original and altered amino acid residues were relatively high, with a Grantham value of >50, as shown in Table 3 (Grantham, 1974). In spite of their high conservation rates, the functional effects of the m.3472T > C transition (Phe56Leu) in the MT-ND1 gene and the m.9604A > G transition (Asn133Ser) in the MT-CO3 gene would seem to be small, because the physicochemical properties are similar between the original and altered amino acid residues, with a Grantham value of <50 (Table 3). 3.3. Novel heteroplasmic variants in the MT-RNR1, MT-TW, and MTND5 genes of mtDNA from schizophrenic patients In addition to the homoplasmic mtDNA variants, we detected three novel heteroplasmic variants: m.1227G > A in the MT-RNR1 gene, m.5578T > C in the MT-TW gene, and the m.13418G > A in MT-ND5 gene on the sequence electropherograms (Fig. 3). These heteroplasmic variants were detected only in each of the distinct schizophrenic patients. None of these three replacements were reported in the three databases described above. The mutation load of the m.1227G > A substitution detected in patient SPsq0066 was approximately 60%, as determined by PCRRFLP (data not shown). According to the core secondary structure model for mammalian MT-RNR1 (12S rRNA) provided by Springer et al. (1996), this substitution disrupts the Watson–Click base pair (G  C ? A # C) in the stem portion of the 29th stem-loop structure (Fig. 4a). This stem-loop structure is conserved among SSU rRNAs (small subunit rRNA) of Archaea, Bacteria, and most Eucarya (Wuyts et al., 2002). The Watson–Click base pair at this structure is highly conserved among species (Gutell et al., 2002), suggesting that the disruption of Watson–Click base pair can result in a certain dysfunction of the MT-RNR1 molecule. To verify the heteroplasmy and quantify the mutation loads of the m.5578T > C substitution in the MT-TW gene, we conducted a TA cloning assay. The mutation load of the m.5578T > C transition in patient SPsq0071 was approximately 36%. As shown in Fig. 4b, nucleotide position 5578 is located next to the 30 -terminus of the MT-TW molecule; and this position is strictly conserved in all of the 149 mammalian species examined, according to the Mamit-tRNA database (http://w3appli.u-strasbg.fr/mamit-trna/Summary.asp). To verify the heteroplasmy and quantify the mutation load of the m.13418G > A (Gly361Glu) replacement in the MT-ND5 gene,

D4a1

SP0020

15301 10873 10398 9540 8701

N

16209 12771 4958 4386 2772 2626

M7a

16324 14364 5899insC

M7a1

11084 11017 522-523d

M7a1a

11722 9299

M7a1a1

9824 6455

M7

16192

M7b1

16319 10397 5330†

15217 504

16519 16129 14979 8473 3206 152

SP0064

D4b1 16319 15951 15440 10181

16287 6689 431

D4b1b

3472*

SP0089

M7b’c

16297 12405 7853 7684 6680 5460 5351 4164 4048 150

M7b

16129 12811

16319 16297 152

204

L3

16298 16189 10345

M7b2

16243 16183C 14155 12361 523insCA

4071 199

16399 10084 6410 4859

D4b1b1

12172

D4b1b1a

11257

D4b

13392† 12795† 10692†

SP0038

8020 522-523d

14560C 8281-8289d

D4b1b2

16093 14584 7043 5460 209

SP0034

16519

14992†

9824A 8964 1382C

D4b2

9296 194

D4b2b

14605

15524

Z2

Z

16298 15487T 8584 7196A 4715

249d

CZ

16260 16185 15784 9090 6752 152

M8

16126

15184 14668 8188 3145

12681† 6710† 16183C 523insCA 8521

16319 14470 8684 6179

M8a

16184 2835

M8a2

13050 6671 4670

M8a2b

12921† 152

15043 14783 10400 489

M

6167G 5147 482

16519 15497 15323 8200

G1

7867

G1a

16325 15860 150

G1a1

11914 4793

G1a1a

1299

G1a1a2

12144# 10550†

16362 14569 5108 4833 709

G

13563 5601

G2

16278 14200 9575 9377 7600

G2a

16227

G2a1

16519 16195 16194 16189

G2a1c

16274 16194C 16183C 6962

9431 8419 8383

D4c2

9536 3316

D4e1

15924 14470 5964 94

D4e1a

9495 4959 214

D4e1a2

334

SP0072 SP0073

16240C 150

11215

D4e

13716

D4e2a

573insC

16093 8945*

SP0035 SP0016

15874

D4e2

16093 12684† 13737 11764 11287

SP0092 SP0011 SP0024 SP0049

16519 16497 11013A*

16519 11255 8764 7270 4538 2706

D4f

4491

M9

3394 153

M9a’b

16316 16234 14308 9242 1041

M9a

11963 153

M9a1

6371†

16183C 16182C 12026 11944 752

D5a

16519 12613 D5a2a 9377 6185 5899insC 16164 3337 16092 13278 D5a1 1310 44insC 16390 D5a2 13708 3496T 16266 68 1438 13437

D5b1a

9180

6253

D5b1

16362

16189

D5’6

D6

16274 13194 7879 7424 6701

12654 3714 1719 709

15622 14927 4216 4200T 152 151

D5c

D

15106 8014† 2178 198 195

5178A 4883

8895† 8860 8618 8020

10397 5301 1107 150

3759

16519 16390 16223 16190 16093 13105 9554 3546 217 182 146

D5

15724 5153 1048 681 456

D5b

16216 7220 2416

D5b1b1

16519 16093 146

D5b1b

16167 16519 6749†

D5a’b

12398* 522-523d

D4g1

D4g 13104

13512 9004

16368 3535 1310

D4l

10427 9355

SP0067 SP0047 SP0068

D4g1b

12684† 146

SP0019

16278 15518 8701 4343 573insC

SP0025 SP0079

16290 10646 195

D4o

16519 16319 16274 16249 16183 13500 9833 2330

D4 14668 8414 3010

11696

D4j

16311

16184

16525 16157 16398 15139 16129† 16293 13656† 13632 5580 13356† 13410 5578 12630 1709 † 152 3753 † 11218

SP0006 SP0069 SP0021 SP0022 SP0071

6227† 182

16399 16037

5438†

SP0033 SP0046 SP0055

SP0051 SP0053 SP0070 SP0057 SP0077 SP0078 SP0002 SP0005 SP0040 SP0091

16245

SP0026

2766

D4c1

16223 9755 3391 207 199 194 191insA

D4c

629 394#

SP0017 SP0088

D4c1a

12509* 11914 188#

SP0010 SP0058

SP0086

D4b2a

10104 8251

D4b2a2

16189 14287

D4b2a2a

16209

SP0007 SP0044

D4b2b1

3866* 150

SP0018

SP0076 SP0085 SP0030 SP0056 SP0081 SP0041 SP0093 SP0094

M7b1’2

16311 10586 7961

SP0012

12957

16249 5466

D4a

D4a2

10005 8296 3531

D4a2a

SP0042

D4a3

SP0027

13651

16266 15440 13418 10601† 7912

SP0084

SP0043

10410

15403† 10084

SP0039

D4a1b

7085†

SP0015

16286 16129 15927 16262insC 15766T† 10373 15586 5339† 2523#

SP0031

15314 5261

D4a1a

12235# 185

SP0090

388 H. Ueno et al. / Mitochondrion 9 (2009) 385–393

Fig. 1. The mtDNA phylogenetic tree for 93 schizophrenic patients displaying the different subsets of macrohaplogroup M and N. The A/C stretch length polymorphism in region 303-315, known to be hypervariable, was disregarded for tree reconstruction. Suffixes A, C, G, and T refer to transversions; ‘d’ means deletion; and ‘ins’ indicates an insertion event (the exact number of the inserted nucleotide(s) was disregarded). Recurrent mutations are underlined. Asterisks () indicate rare non-synonymous substitutions in the mitochondrial protein-coding regions that were detected only in schizophrenic patients but not in the 784 controls. Number signs (#) indicate rare substitutions in the mitochondrial RNA genes or non-coding region that were detected only in schizophrenic patients but not in the 784 controls. Daggers ( ) indicate rare synonymous substitutions in the mitochondrial protein-coding regions that were detected in schizophrenic patients but not in the 784 controls. SP indicates a schizophrenic patient.

we conducted both PCR-RFLP and TA cloning assays. The mutation load in patient SPsq0084 was approximately 74%. As shown in Fig. 4c, amino acid position 361 is located in the outer membrane side of the p. MT-ND5 subunit. The Gly361 residue was highly con-

12880 5773

A5a1a

16399 11914

SP0074

14944

A5a1

10801

SP0009 16354 13889* 5894 4736

A5a1a1

SP0059

SP0036

SP0052

7664

B4b1a1a

16223 10496† 8723* 189

SP0061

16187 11647 4655 2156insA 152

A5a

8628

16189 3397

SP0065

15535 827

B4b’d

13590 4820 499

B4b

16136

B4b1

6413 6023 207

B4b1a

16284 15236 8206 4117 2831 202 199

B4b1a1

8510* 204

SP0095

11536 8563

A5

13461

A5a2

SP0075

16182C

A

16213 16129 12816 522-523d

A5c

12816

16319 16290 8794 4824 4248 1736 663 522-523d 235 152

16235 16126 1709 961

A5b

16519 16235 10637 9545 965insC 152

B4c

16311 3497

B4c1

9711 8962 8781† 2857

SP0028

15346 1119

SP0037 11296† 9604*

SP0062

14133 10310 709

B4c1a

16086 8844

B4c1a1

16129 5592

B4c1a1a

SP0023

16217

B4

8843* 152

SP0080

16291 14016 9300*

SP0050

8281-8289d

B

15941 13629 10398 5441 214 195 150

B4c1c

16519 16304 16280 16217 14178

SP0063

15257 5553G 4688A

SP0029

B5b

B5

16243 15927 15851 15662 15508 15223 12361 8829 1598 204

16261 16257A 12372 12358 5231 150

B5b2

16399T 16311 13879 8027* 7424 146

15067 961

N9a2

15301 10873 10398 9540 8701

N

5417

N9

8989

SP0048

16172

16463 16234 16111 15850 4895

SP0054

16497 965insC

N9a

16319 8902* 8419

SP0083

16519 16189 16183C

16140 10398 9950 8584 709 522-523d

B5b1 14470 11146 8784 960insC

B5b2a

16319 16318T 16223 12192 11914 199 103 16291 11437 5744 131

16182C 195

SP0096

4500* B5b1b

SP0087

16209 16189 15883 3729

N9a5

13132A* 12469 5460 1943

SP0082

16311 13681 12950 11061 10398 10031 8278insC 8277 709 189 185

R11

10978

R11a

16365# 14696 12358 9071* 5301 5186C* 4706†

SP0032

16519 16189 14893 13183 11016 10607 5147

N9b

12501

N9b1

16183C 14356† 14311 12561† 9128 3796* 249d 207 204 152 94

SP0001

14766 11719 73

HV

7028 2706

H

4769 1438

H2

15326 8860 750 263

Revised CRS

16359 16355 15496 15024 15022 13749 13260 12618 10007 5587 3027 1227

SP0066

16223 12705

R

16519 12882 12406 10609 6962 522-523d

F1

16189 16183C 16182C

14629 8251 465#

SP0008

16311 16249 16232A 14476 12633 10976 152

F1b

16344 16129 5147 4732

F1b1

16304 13928C 3970

R9

10310 6392 249d

151

SP0045

F

15954 5049

F1b1a

4705

14103A F1b1a1

F1b1a1a

SP0060

15670 12630 5263

F4

16399 16362 16207 13602 12153 10915 3290 146

F4a

16497 12408 12396 11038 7861 1520 207 152

F4a1

14693

SP0014

H. Ueno et al. / Mitochondrion 9 (2009) 385–393 389

Fig. 1 (continued)

served in 58 of the 61 mammalian species examined. Although a serine residue in Erinaceus europaeus (western European hedgehog) and an asparagine residue in Mus musculus (house mouse) and Rattus norvegicus (Norway rat) existed at this position, no

390

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Table 2 MtDNA haplogroup distribution in schizophrenic patients compared with that of 784 Japanese control subjects. Haplogroups

D4 A B N9a M7b D5 F M7a G2 G1 Others a

Schizophrenic patients

Japanese controls

Number

%

Number

%

35 9 8 6 6 6 5 3 2 2 8

39 10 8.9 6.7 6.7 6.7 5.6 3.3 2.2 2.2 8.9

278 55 104 39 36 31 48 52 36 28 77

35 7.0 13 5.0 4.6 4.0 6.1 6.6 4.6 3.6 9.8

P valuea

0.56 0.29 0.32 0.45 0.43 0.26 1.00 0.36 0.42 0.76 – Fig. 2. Locations in the secondary structures of the p.MT-ATP6 subunit of Ile106Thr, Ala126Thr, and Met140Thr homoplasmic replacements.

P value was calculated by Fisher’s exact probability test.

acidic residue such as glutamate was found there among the 61 mammalian species examined. Additionally, we detected a heteroplasmic m.12613G > A substitution in only one schizophrenic patient (SPsq0070); however, this substitution was registered as a polymorphism in both the mtDB and the mtSNP database. 4. Discussion In the present study, we detected three different novel heteroplasmic variants, i.e., m.1227G > A in the MT-RNR1 gene, m.5578T > C in the MT-TW gene, and m.13418G > A in the MTND5 gene among 93 patients with schizophrenia (Fig. 3). All three patients bearing the mutation had no apparent neuromuscular symptoms and were not inspected for further biological or neurological characteristics including their plasma lactate level or examined by muscle biopsy or magnetic resonance spectroscopy (MRS). Patient SPsq0066, with m.1227G > A, was a 49-year-old woman diagnosed as having schizophrenia at age 41. Patient SPsq0071, with m.5578T > C, was a male with schizophrenia (disorganized

type), whose age at onset is unknown; and patient SPsq0084, with m.13418G > A, was diagnosed as having schizophrenia (residual type) at age 49. Although the functional role of the m.1227G > A variant remains unclear, we can predict the functional effects of the other two variants (m.5578T > C and m.13418G > A). The novel m.5578T > C replacement in the MT-TW gene was located next to the 30 -terminus of the gene (Fig. 4b). Two pathogenic mutations in the tRNA Leucine 1 (MT-TL1) gene (m.3303C > T and m.3302A > G) were also located adjacent to the 30 -terminus of the MT-TL1 molecule. These mutations were earlier suggested to affect the tRNA end processing, such as cleavage of tRNA 30 -trailers or synthesis of the CCA terminus (Levinger et al., 2004). It is possible that 5578T is also functionally important and that the m.5578T > C replacement might have a pathogenic nature associated with the development of schizophrenia in this patient. Interestingly, some preceding studies suggested that mutations in the MT-TW gene were often associated with dementia or other neuropsychiatric disturbances in the central nervous system (Silvestri et al., 2000; Santorelli et al., 1997; Nelson et al., 1995).

Table 3 Non-synonymous homoplasmic substitutions in the mitochondrial protein-coding regions detected in schizophrenic patient but not in Japanese control subjects. Gene

Nucleotide substitution (amino acid change)

Individual

AA conservation among 61 mammalian species

Grantham value

MT-ND1 MT-ND1 MT-ND1 MT-ND2 MT-ND2 MT-CO2 MT-ATP8 MT-ATP6 MT-ATP6 MT-ATP6 MT-ATP6 MT-ATP6 MT-CO3 MT-CO3 MT-ND4 MT-ND5

3472T > C (Phe56Leu) 3796A > G (Thr164Ala) 3866T > C (Ile187Thr) 4500T > C (Ser11Pro) 5186A > C (Trp239Cys) 8027G > A (Ala148Thr) 8510A > G (Lys49Glu) 8723G > A (Arg66Gln) 8843T > C (Ile106Thr) 8902G > A (Ala126Thr) 8945T > C (Met140Thr) 9071C > T (Ser182Leu) 9300G > A (Ala32Thr) 9604A > G (Asn133Ser) 11013C > A (Ser85Tyr) 12398C > T (Thr21Ile)

60 (98%) 40 (66%) 54 (89%) 16 (26%) 8 (13%) 10 (16%) 7 (11%) 31 (51%) 56 (92%) 59 (97%) 60 (98%) 14 (23%) 14 (23%) 61 (100%) 30 (49%) 11 (18%)

22 58 89 74 215 58 56 43 89 58 81 145 58 46 144 89

MT-ND5

12509A > G (Asp58Gly)

MT-ND5 MT-ND5

13132C > A (Leu266Met) 13889G > A (Cys518Tyr)

SPsq0089 SPsq0001 SPsq0018 SPsq0087 SPsq0032 SPsq0054 SPsq0095 SPsq0061 SPsq0080 SPsq0083 SPsq0035 SPsq0032 SPsq0050 SPsq0037 SPsq0033 SPsq0057 SPsq0010 SPsq0058 SPsq0082 SPsq0009

6 (9.8%) 49 (80%) 1 (1.6%)

94 15 194

Amino acid (AA) sequences of mitochondrial protein-coding genes from 61 mammalian species were obtained from the mtSNP database. The Grantham value was calculated from the physicochemical differences between the original and altered amino acid residues to evaluate the functional alterations. The average of the Grantham values of 20  19/2 = 190 different amino acid replacements is 50. SP, schizophrenic patient; ND, NADH: ubiquinone oxidoreductase subunit; CO, cytochrome c oxidase subunit; ATP, ATP synthase subunit.

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391

Fig. 3. Electrofluorograms of three heteroplasmic variants: (a) m.1227G > A in the 12S rRNA (MT-RNR1) gene, (b) 5578T > C in the tRNA tryptophan (MT-TW) gene, and (c) m.13418G > A in the MT-ND5 gene.

In patients with mitochondrial encephalomyopathies (e.g., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes syndrome, so-called MELAS), the percentage of mutant mtDNA is much higher in the affected tissues including brain or skeletal muscles than in the blood cells (Nishigaki et al., 2003b). Although the mutation load of m.5578T > C was not so high (approximately 36%) in the blood, it is therefore possible that the percentage of the mutation in the brain of the patient was higher than that in the blood cells. As we could not obtain DNA samples from family members of this patient, we were unable to confirm whether the mutation load of m.5578T > C co-segregated with the presence or absence of schizophrenic symptoms in this family. The novel m.13418G > A replacement causes the Gly361Glu replacement in the matrix side of the p.MT-ND5 subunit. The transmembrane topology of the NuoL subunit (p.MT-ND5 homologue) from Rhodobacter capsulatus Complex I has been experimentally determined by Mathiesen and Hagerhall (2002). According to their model, this Gly361Glu replacement is located in the large domain in the mitochondrial matrix side, which corresponds to the helices X and XI of NuoL of R. capsulatus (Fig. 4 of the reference Mathiesen and Hagerhall, 2002). Because the Gly361 (corresponding to Gly376 of NuoL of R. capsulatus) is highly conserved among NuoL/NuoM/NuoN, it is plausible that the amino acid replacement Gly361Glu can result in dysfunction of the p.MT-ND5 subunit of complex I. This domain contains many conserved sequences and seems to be of functional importance (Mathiesen and Hagerhall,

2002). When Gly361 is replaced by the acidic residue Glu, the altered electrostatic interaction between the replaced Glu361 and adjacent residues may result in dysfunction of this subunit (Fig. 4c). Because the patient with m.13418G > A (ID: SPsq0084) had no family history of schizophrenia, it is possible that this nucleotide change is a de novo mutation or that the mutation load in other family members might be low. In addition to the heteroplasmic mtDNA mutations in the patients, certain rare homoplasmic variants can be slightly deleterious and may be associated with schizophrenia. Although it is difficult to identify the functional effects of homoplasmic variants, we can speculate that m.8843T > C, m.8902G > A, and m.8945T > C in the MT-ATP6 gene might affect the function of the p.MT-ATP6 subunit, not only because of their high conservation rate but also because the physicochemical differences between the wild-type and the altered amino acids were relatively large, with a Grantham value of >50 (Table 3). It also remains to be elucidated whether these replacements contribute to the susceptibility to schizophrenia. Recent studies on mitochondrial abnormalities in postmortem brains from subjects with neurological disorders (Kung and Roberts, 1999) have prompted us to reappraise the contribution of common or rare variants in mtDNA to the risk of schizophrenia. The analysis of the distribution of mitochondrial haplogroups in Israeli schizophrenic patients compared with that of their healthy fathers (202 pairs) revealed an over-representation of the mtDNA lineage cluster HV in the patients (P = 0.01), with an increased rel-

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Fig. 4. Locations in the predicted secondary structures of: (a) MT-RNR1 molecule of heteroplasmic substitution m.1227G > A, (b) MT-TW molecule of heteroplasmic substitution m.5578T > C, and (c) p.MT-ND5 subunit of Gly361Glu heteroplasmic replacement. In ‘‘a,” the location of m.1227G > A is shown in the core secondary structure model for mammalian 12S rRNA provided by Springer and Douzery (1996). In ‘‘b,” the substitution m.5578T > C is located at the 30 terminal region of the MT-TW molecule. In ‘‘c,” the Gly361Glu replacement is located in a large domain corresponding to helices X and XI of the NuoL subunit of Complex I of R. capsulatus. Amino acid sequences show inter-species similarity of the p.MT-ND5. The Gly361 in patient SPsq0084 is highlighted with a bold letter and is compared to human p.MT-ND5 subunit and the NuoL subunit of Complex I of R. capsulatus.

ative risk (odds ratio) of 1.8 among Israeli Arabs (Amar et al., 2007). However, our data suggest that none of the 10 major Japanese haplogroups were significantly associated with schizophrenia (Table 2). It seems that mitochondrial haplogroup distribution associated schizophrenia has a different tendency among ethnic populations. Further studies with larger number of samples will be needed to clarify this issue. Martorell et al. (2006) determined the entire mtDNA of six schizophrenic patients with an apparent maternal transmission of the disease. They identified three novel missense variants, i.e., m.7750C > A (p.MT-CO2: Ile55Met), m.8857G > A (p.MTATP6: Gly111Ser), and m.12096T > A (p.MT-ND4: Leu446His). This last variant was a heteroplasmic mutation that was detected in five of the six mother-offspring patient pairs, which variation is a non-conservative substitution in the p.MT-ND4 subunit of complex I. The contribution of these mutations to the pathogenesis of schizophrenia needs further validation (Bandelt et al., 2007). Kazuno et al. (2005) sequenced the entire mtDNA sequences of seven patients with various psychotic disorders including schizoaffective disorder. Although none of them had known

mtDNA mutations pathogenic for mitochondrial encephalopathy, the authors described that two of the seven patients belonged to subhaplogroup F1b1a and suggested that this haplogroup might be a good target for an association study of ‘atypical psychosis.’ These preceding studies had some limitations such as small sample size or focus only on specific mtDNA regions or mutations. However, our study is the most extensive one to date in two aspects: first, we determined the entire mtDNA sequences from as many as 93 schizophrenic patients. Second, we investigated the mtDNA variants in three different categories: (1) common ancient polymorphisms that are associated with each haplogroup, (2) rare homoplasmic variants that are specific to each maternal lineage, and (3) heteroplasmic mutations that are potentially pathogenic. There is no doubt that further research is needed to elucidate the contribution of mtDNA variants and mutations to schizophrenia. We believe that our findings will enhance the understanding of this disorder and encourage new directions in schizophrenia research, which in turn, should have an impact on treatment approach, diagnosis, and disease prevention of schizophrenia and related syndromes.

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Acknowledgments This work was supported in part by the Support Project for Database Development from the Japan Science and Technology Corporation (to M.T.); a Grant-in-Aid for Scientific Research [C18590317 to Y.N., and C2-10832009 and A (2)-15200051 to M.T.], a Grant-in-Aid for Young Scientists (B-18700541 to N.F.), and a Grant-in-Aid for Exploratory Research (20650113 to N.F.) from the Ministry of Education, Culture, Sports, Science and Technology; by a grant from the Third-Term Comprehensive 10-year Strategy for Cancer Control (to M.T.); by Grant 20B-13 from the program Research Grants for Nervous and Mental Disorders of the Ministry of Health, Labour, and Welfare (to M.T.); and by grants for scientific research from the Kao Research Council for the study of Healthcare Science (to Y.N.) and from the Takeda Science Foundation (to Y.N. and to M.T.) and Sankyo Foundation of Life Science (to Y.N.). References Alexe, G., Fuku, N., Bilal, E., Ueno, H., Nishigaki, Y., Fujita, Y., Ito, M., Arai, Y., Hirose, N., Bhanot, G., Tanaka, M., 2007. Enrichment of longevity phenotype in mtDNA haplogroups D4b2b, D4a, and D5 in the Japanese population. Hum. Genet. 121, 347–356. Allen, N.C., Bagade, S., McQueen, M.B., Ioannidis, J.P., Kavvoura, F.K., Khoury, M.J., Tanzi, R.E., Bertram, L., 2008. Systematic meta-analyses and field synopsis of genetic association studies in schizophrenia: the SzGene database. Nat. Genet. 40, 827–834. Amar, S., Shamir, A., Ovadia, O., Blanaru, M., Reshef, A., Kremer, I., Rietschel, M., Schulze, T.G., Maier, W., Belmaker, R.H., Ebstein, R.P., Agam, G., Mishmar, D., 2007. Mitochondrial DNA HV lineage increases the susceptibility to schizophrenia among Israeli Arabs. Schizophr. Res. 94, 354–358. Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H., Coulson, A.R., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J., Staden, R., Young, I.G., 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465. Andrews, R.M., Kubacka, I., Chinnery, P.F., Lightowlers, R.N., Turnbull, D.M., Howell, N., 1999. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet. 23, 147. Bandelt, H.-J., Olivieri, A., Bravi, C., Yao, Y.-G., Torroni, A., Salas, A., 2007. ‘Distorted’ mitochondrial DNA sequences in schizophrenic patients. Eur. J. Hum. Genet. 15, 400–402. author reply, 402–404. Bilal, E., Rabadan, R., Alexe, G., Fuku, N., Ueno, H., Nishigaki, Y., Fujita, Y., Ito, M., Arai, Y., Hirose, N., Ruckenstein, A., Bhanot, G., Tanaka, M., 2008. Mitochondrial DNA haplogroup D4a is a marker for extreme longevity in Japan. PLoS ONE 3, e2421. Cavelier, L., Jazin, E.E., Eriksson, I., Prince, J., Bave, U., Oreland, L., Gyllensten, U., 1995. Decreased cytochrome-c oxidase activity and lack of age-related accumulation of mitochondrial DNA deletions in the brains of schizophrenics. Genomics 29, 217–224. Doi, N., Hoshi, Y., 2007. Persistence problem in schizophrenia and mitochondrial DNA. Am. J. Med. Genet. B Neuropsychiatr. Genet. 144B, 1–4. Fuku, N., Park, K.S., Yamada, Y., Nishigaki, Y., Cho, Y.M., Matsuo, H., Segawa, T., Watanabe, S., Kato, K., Yokoi, K., Nozawa, Y., Lee, H.K., Tanaka, M., 2007. Mitochondrial haplogroup N9a confers resistance against type two diabetes in Asians. Am. J. Hum. Genet. 80, 407–415. Grantham, R., 1974. Amino acid difference formula to help explain protein evolution. Science 185, 862–864. Gutell, R.R., Lee, J.C., Cannone, J.J., 2002. The accuracy of ribosomal RNA comparative structure models. Curr. Opin. Struct. Biol. 12, 301–310. Karry, R., Klein, E., Ben Shachar, D., 2004. Mitochondrial complex I subunits expression is altered in schizophrenia: a postmortem study. Biol. Psychiatr. 55, 676–684. Kazuno, A.A., Munakata, K., Mori, K., Tanaka, M., Nanko, S., Kunugi, H., Umekage, T., Tochigi, M., Kohda, K., Sasaki, T., Akiyama, T., Washizuka, S., Kato, N., Kato, T., 2005. Mitochondrial DNA sequence analysis of patients with ‘atypical psychosis’. Psychiatr. Clin. Neurosci. 59, 497–503. Kong, Q.-P., Bandelt, H.-J., Sun, C., Yao, Y.-G., Salas, A., Achilli, A., Wang, C.-Y., Zhong, L., Zhu, C.-L., Wu, S.-F., Torroni, A., Zhang, Y.-P., 2006. Updating the East Asian mtDNA phylogeny: a prerequisite for the identification of pathogenic mutations. Hum. Mol. Genet. 15, 2076–2086. Kong, Q.-P., Salas, A., Sun, C., Fuku, N., Tanaka, M., Zhong, L., Wang, C.-Y., Yao, Y.-G., Bandelt, H.-J., 2008. Distilling artificial recombinants from large sets of complete mtDNA genomes. PLoS ONE 3, e3016. Kung, L., Roberts, R.C., 1999. Mitochondrial pathology in human schizophrenic striatum: a postmortem ultrastructural study. Synapse 31, 67–75. Levinger, L., Morl, M., Florentz, C., 2004. Mitochondrial tRNA 30 end metabolism and human disease. Nucleic Acids Res. 32, 5430–5441.

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