Gene expression analysis in schizophrenia: Reproducible up-regulation of several members of the apolipoprotein L family located in a high-susceptibility locus for schizophrenia on chromosome 22 Michael L. Mimmack*†, Margaret Ryan†‡, Hajime Baba§, Juani Navarro-Ruiz¶, Shuji Iritaniʈ, Richard L. M. Faull**, Peter J. McKenna††, Peter B. Jones††, Heii Arai§, Michael Starkey‡, Piers C. Emson*, and Sabine Bahn*††‡‡ *Department of Neurobiology, Babraham Institute, Cambridge CB2 4AT, United Kingdom; ‡United Kingdom Human Genome Mapping Project Resource Centre, Hinxton, Cambridge CB10 1SB, United Kingdom; ††Department of Psychiatry, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom; **Department of Anatomy, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92019, Auckland, New Zealand; ʈDepartment of Psychiatry, Tokyo Metropolitan Matsuzawa Hospital, 156-0057 Tokyo, Japan; §Department of Psychiatry, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, 113-8421 Tokyo, Japan; and ¶Department of Morphological Sciences and Psychobiology, School of Medicine, University of Murcia, 30120 Murcia, Spain Communicated by Michael J. Berridge, Babraham Institute, Cambridge, United Kingdom, February 5, 2002 (received for review October 8, 2001) We screened a custom-made candidate gene cDNA array comprising with lipids that are of vital importance to normal cell functioning. 300 genes. Genes chosen have either been implicated in schizophre- Lipids are used to generate energy, are building blocks for biomem- nia, make conceptual sense in the light of the current understanding branes, are essential for the synthesis of numerous lipophilic of the disease, or are located on high-susceptibility chromosome molecules (e.g., steroid hormones and vitamin E), and play an locations. The array screen using prefrontal cortex tissue from 10 important role in cell signaling and antioxidative mechanisms (6, 7). schizophrenia and 10 control brains revealed robust up-regulation of Most lipoproteins are expressed in many tissues and cell types, apolipoprotein L1 (apo L1) by 2.6-fold. The finding was cross-vali- including the brain and cerebrospinal fluid (6). Apo L belongs to dated in a blinded quantitative PCR study using prefrontal cortex the high density lipoprotein family that plays a central role in tissue from the Stanley Foundation brain collection, Bethesda, MD. cholesterol transport (8). The cholesterol content of membranes is This collection consists of 15 schizophrenia, 15 bipolar disorder, 15 important in cellular processes such as modulating gene transcrip- major depression, and 15 control individuals, all 60 brains being tion and signal transduction both in the adult brain and during well-matched on conventional parameters, with antipsychotic drug neurodevelopment (7). exposure in the schizophrenia and bipolar disorder groups. Signifi- cant up-regulation of apo L1 gene expression in schizophrenia was Methods confirmed. Using quantitative PCR, expression profiles of other mem- Tissue Collection. Fresh-frozen prefrontal cortex tissue was obtained bers of the apo L family (apo L2–L6) were investigated, showing that from the Stanley Foundation brain collection and neuropathology apo L2 and L4 were highly significantly up-regulated in schizophrenia. consortium, Bethesda, MD, as well as from a collection of schizo- Results were then confirmed in an independent set of 20 schizophre- phrenia and control brains from Japan (Department of Psychiatry, nia and 20 control brains from Japan and New Zealand. Apo L proteins Tokyo Metropolitan Matsuzawa Hospital) and from New Zealand belong to the group of high density lipoproteins, with all six apo L (New Zealand Neurological Foundation Human Brain Bank, Uni- genes located in close proximity to each other on chromosome 22q12, versity of Auckland, School of Medicine). The Stanley Foundation a confirmed high-susceptibility locus for schizophrenia and close to brain collection consists of well-matched prefrontal cortex tissue the region associated with velocardiofacial syndrome that includes symptoms of schizophrenia. derived from 15 schizophrenia, 15 bipolar disorder, 15 major depression, and 15 control individuals (9). The New Zealand͞Japan collection consists of corresponding prefrontal cortex tissue from chizophrenia is a major public health problem. Typically, psy- 20 schizophrenia and 20 matched controls. For all cases, tissue was Schosis begins in early adulthood and interferes with education, collected from patients who had previously been diagnosed accord- employment, and social functioning. A hereditary contribution has ing to DSM-IIIR (33) or DSM-IV (34). The Stanley Foundation been established beyond doubt, and neuroimaging and neuropatho- brain collection is well-matched for age, pH, sex, race, side of the logical studies show subtle brain abnormalities (1–3). The dorsal brain, and postmortem interval, the details of which have been prefrontal cortex has been particularly implicated in the patho- described by Torrey et al. (9). In brief, the mean age in the physiological dysfunction in schizophrenia (4, 5). schizophrenia group was 44.2 (range 25–62) years, 42.3 (range The sequencing of the human genome undoubtedly will open 25–61) years for bipolar disorder, 46.4 (range 30–65) for major new exciting avenues for research into complex disorders such as depression, and 48.1 (range 29–68) for normal controls. The sex schizophrenia. Furthermore, the availability of novel molecular ratio was nine males and six females in all groups, and the average profiling techniques such as microarrays and differential display– postmortem interval for the four groups ranged between 23.7 and PCR now allow a global approach to screening for abnormalities in gene expression. Such approaches have the benefit that no assumption has to be made as to whether pathology is caused Abbreviations: apo, apolipoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; primarily by genetic and͞or environmental factors, as any caus- TNF-␣, tumor necrosis factor ␣. ative effect will inevitably lead to changes in gene expression. †M.L.M. and M.R. contributed equally to this work. For this study, we designed a candidate gene cDNA array to ‡‡To whom reprint requests should be addressed. E-mail: [email protected]. investigate changes in gene expression in the prefrontal cortex in The publication costs of this article were defrayed in part by page charge payment. This schizophrenia. We found robust and reliable changes in expression article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. of apolipoprotein (apo) L1, L2, and L4. Lipoproteins provide cells §1734 solely to indicate this fact. 4680–4685 ͉ PNAS ͉ April 2, 2002 ͉ vol. 99 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.032069099 Downloaded by guest on October 1, 2021 33.7 h (see ref. 9 for more details). All 60 tissue samples from the oligo(dA80) for 2.5 h at 42°C in an attempt to prevent hybridization Stanley Foundation were analyzed blind to diagnostic status with of poly(T) sequences to poly(A) tails of immobilized cDNAs. the code being retained by the foundation until analysis was Hybridization was for 48 h at 42°C in 9.5 ml of hybridization buffer completed. The New Zealand͞Japan brain collection was matched (Ultrahyb, Ambion). Arrays were washed sequentially in 2ϫ SSC, for age, sex, and postmortem interval. Whereas brains from the 0.1% SDS at room temperature (2 ϫ 5 min), 2ϫ SSC, 0.1% (wt͞vol) Stanley Foundation collection were predominantly young to SDS at 42oC(2ϫ 5 min), and 0.1ϫ SSC, 0.1% (wt͞vol) SDS at 42oC middle-aged, the New Zealand͞Japan collection consists of mainly (2 ϫ 15 min) and exposed before scanning with the Typhoon 8600 elderly and chronically (mostly lifelong) hospitalized individuals. variable mode imager (Amersham Pharmacia). To normalize for For the schizophrenia group the mean age was 65.3 years (range variation in robotic spotting each membrane was stripped and 43–85 years) and for controls 62.7 years (range 42–84 years); the sex rehybridized with 5 ng of 33P-dATP-labeled pBluescript II SK(Ϫ) ratio was 15 males and five females for the schizophrenia group and vector. After image acquisition, hybridization signals were quanti- 17 males and three females for controls. Postmortem interval fied by using ArrayVision (Image Research, St. Catherines, Cana- averages were 8.1 h (range 2–18 h) for schizophrenia and 12.6 h da). Signal processing was subsequently performed by using Blue- (range 2–25 h) for controls. script vector data to correct for variation in the relative amount of DNA present at equivalent positions on different arrays. Variations RNA Extraction and cDNA Synthesis. Total RNA was extracted from in labeling, hybridization, washing, and duration of phosphor screen postmortem prefrontal cortices of schizophrenia, bipolar, de- exposure for different array hybridizations were normalized against pressed, and control brains by using the Tri-reagent RNA plant spike expression profiles (A. thaliana cytochrome c554). extraction protocol (Sigma) in conjunction with a mechanical Quantified data were expressed as absolute gene expression levels homogenizer (Thermohybaid, Middlesex, U.K.). Typical yields and were comparable within all RNA samples. were 1 ␮g of total RNA per 1 mg of tissue. RNA sample concentrations were determined by UV spectrophotometry (av- Real-Time Quantitative PCR. Real-time PCR was performed by Ͼ erage OD260/280 1.8). RNA integrity was confirmed by