Journal of Human Genetics (2011) 56, 869–872 & 2011 The Japan Society of Human Genetics All rights reserved 1434-5161/11 $32.00 www.nature.com/jhg SHORT COMMUNICATION Analysis of complete mitochondrial genomes of patients with schizophrenia and bipolar disorder Cinzia Bertolin1,4, Chiara Magri2,4, Sergio Barlati2, Andrea Vettori1, Giulia Ida Perini3, Pio Peruzzi3, Maria Luisa Mostacciuolo1 and Giovanni Vazza1 The present study aims at investigating the association between common and rare variants of mitochondrial DNA (mtDNA), and increased risk of schizophrenia (SZ) and bipolar disorder (BPD) in a cohort of patients originating from the same Italian population. The distribution of the major European mtDNA haplogroups was determined in 89 patients and their frequencies did not significantly differ from those observed in the Italian population. Moreover, 27 patients with high probability of having inherited the disease from the maternal side were selected for whole mitochondrial genome sequencing to investigate the possible presence of causative point mutations. Overall, 213 known variants and 2 novel changes were identified, but none of them was predicted to have functional effects. Hence, none of the sequence changes we found in our sample could explain the maternal component of SZ and BPD predisposition. Journal of Human Genetics (2011) 56, 869–872; doi:10.1038/jhg.2011.111; published online 13 October 2011 Keywords: bipolar disorder; mtDNA variants; schizophrenia INTRODUCTION segregation of SZ or BPD, as well as the co-segregation of both phenotypes, have Schizophrenia (SZ) and bipolar disorder (BPD) are among the top ten been observed. This is in agreement with the hypothesis of a genetic overlap causes of disability worldwide.1 Despite extensive genetic and phar- between SZ and BPD; in this study, we hence considered both SZ and BPD cases. macological studies, the etiology and pathophysiology of these mental Overall, the sample included a total of 89 patients belonging to different maternal disorders are still unknown, and the nuclear susceptibility loci lineages; among them, 35 were sporadic and the other 54 were extracted from 41 complex pedigrees (for further details see Supplementary Materials and Methods). identified so far can explain only a small fraction of the genetic 2 component of these diseases. Mt-Haplogroup analysis and mtDNA genome resequencing The growing body of observations published in the last decade We investigated the distribution of mtDNA haplogroups (mt-hgs) in the 89 SZ points to the involvement of mitochondria in the pathophysiology of and BPD patients by combining restriction fragment length polymorphism psychiatric disorders, including SZ and BPD.3 Several genetic studies analysis, with sequencing of hypervariable sequence I (HVS-I) of the mito- reported association between these diseases and common mitochon- chondrial displacement-loop. The most common European mt-hgs were drial DNA (mtDNA) polymorphisms defining ethnic-specific mito- classified using single-nucleotide polymorphisms previously reported.9 chondrial haplogroups.4–5 Other analyses found new rare variants In addition, a 466-bp fragment encompassing the HVS-I was sequenced in with a putative functional effect6 and a global excess of synonymous all samples. Sequence variants were determined between mtDNA nucleotides substitutions in the dorsolateral prefrontal cortex of SZ patients.7 16001 and 16400 by comparison with the revised Cambridge Reference Sequence (rCRS; GenBank accession no. NC_012920.1). Despite the great interest of these findings, the role of mtDNA in The complete mt-genome was sequenced using the GeneChip Mitochon- the pathogenesis of SZ/BDP remains unclear. drial Resequencing Array 2.0 (MitoChip v.2.0, Affymetrix, Santa Clara, CA, The present study aims at investigating the association between USA) following the manufacturer’s protocol. Sequencing process, data acquisi- common and rare variants of mtDNA and the increased risk of SZ/ tion and statistical analyses were conducted as described in the Supplemen- BPD in a sample of patients originating from the same Italian population. tary Materials and methods. The haplogroup classification was based on the phylogeny proposed by van Oven and Kayser,10 and reported at http:// MATERIALS AND METHODS www.phylotree.org/ (mtDNA tree Build 11). Samples All analyzed subjects originated from the population of Chioggia, a North-East RESULTS AND DISCUSSION Italian town. All patients were diagnosed according to DSM-IV criteria as We used our data to reconstruct the phylogenetic relationships among described previously.8 Most patients belong to complex pedigrees in which the patients’ mtDNAs. The variations found in the HVS-1 region of the 1Department of Biology, University of Padova, Padova, Italy; 2Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, University School of Medicine, Brescia, Italy and 3Department of Neurosciences, University of Padova, Padova, Italy 4These authors contributed equally to this work. Correspondence: Dr G Vazza, Department of Biology, University of Padova, Via G Colombo 3, 35131 Padova, Italy. E-mail: [email protected] Received 19 May 2011; revised 30 August 2011; accepted 1 September 2011; published online 13 October 2011 870 Journal of Human Genetics African/South Asian branch of theregions, N1a they haplogroup, boththough they possess differ from the each(one 16147G other familiar for allele, two and mutationsnow which in in one other the defines samples. coding sporadic) Among our the typical belonging cohort, of we to sequenced the two Italian patients mt-hgsuggests population, that as N1a. variant it Even 8348 had is not diagnostic been for described a until sub-clade of the H1b at position 7666 and 8590 in the public database. These latter arethe two literature and synonymous changes only identified twowere of observed. them Most (Table 2) of are thesetion not variants events, reported have including 148 in been in any already the described coding in region (positions 577–16023), a new sub-clade inside thesuggesting N1a that haplogroup. 151 and 2758 are good candidates to be markers of mt-hgs and peculiar to oursusceptibility mtDNA sample mutations occurring were multiple identified. times in Altogether,The these different new mutations were observed in single patients: no recurrent with the Italian H1b sample reported by Achilli variant m.8348A the same mtDNA sequence.samples Interestingly, (21.2, these 22.1 and samples 16.1), also belonging torelatedness. shared mt-hg In H1b, shared addition exactly todeeper the investigation is twosuggest X2b required that subjects, to the only ascertain two three their subjects other were possible related cryptic in the recent past, and a recurrent mutations. mtDNA variations, ashowever, is many not mitochondrial necessarily incompatible diseases with are thefrequency distribution. due identification of to which would allowthese negative to results could detect be only due to large the changes small size in of our haplogroup sample, sample of Africantwo origins samples shared published themake by mutations the at sequences Gonder peculiar position within 151the the and N1a canonical 2758 lineage. N1a Moreover, with these polymorphism a at position 16248. Such findings identify SZ susceptibility mt-hgs onwere observed. Italian patients. for multiple comparisons andcontrols were no then statistically analyzed significant by differences applying the Bonferroni’s adjustment infrequent occurrence in theshare Italian the population same (2.9%), mtDNAsporadic sequence. cases As revealed hg that X2bthe only is phylogenetic characterized the by tree two an in X2b Figure samples (39.1, 1. 52.1) (for The details see mtDNA Supplementary sequencing Tablesame 1). of mt-hg and the HVS-1 sequence withFigure one 1). of the The other remaining selectedgeneration cases or five with were all-affected siblings sporadic inpedigrees cases, with the either sibship who (Supplementary mother-to-child transmission shared inAmong the more than this one group, 22mtDNA of individuals 27 patients, were searching for familiar susceptibility mtDNA cases mutations. belonging to samples (Table 1). observed when compared with those reported forand three Italian control noto significant mt-hgs; all major differences European mt-hgsHaplogroup were distribution analysis in represented did in not the reveal their a sample diseasefor susceptibility 90% overall of the distributionEuropean genetic haplogroups, that variability is, were observed H, in V, K,89 our U, patients J, sample are T, reported I, (Table in 1). W Supplementary and Table X, 1. accounted The most frequent Considering the 27 sequenced mt-genomes, a total of 215 substitu- The lack of power for association with a mtDNA haplogroup, These findings confirmed a previous study that had failed to All the mtDNA variations detected in the 27 patients are shown in 4 11–13 G with another sample of our collection (4.5) and 14 The frequencies of each mt-hg in patients and We therefore sequenced the complete ATPase6 MtDNA sequencing in SZ and BPD patients and COII et al genes, respectively. et al 17 . 16 and have lost 11 15 ., CBertolin This result 18 However, our data thereby et al Table 1 mtDNA haplogroup distribution in SZ/BPD patients and in three independent samples of the Italian population Fisher’s exact Fisher’s exact Fisher’s Patients Italian CT1a test Italian CT2b test Italian CT3c exact test Hg N¼89 (%) CI (95%) N¼190 (%) CI (95%) P-values N¼1486 (%) CI (95%)