It isIt illegal to post this copyrighted PDF on any website. J ClinPsychiatry 77:5,May 2016 For [email protected]. reprints orpermissions, contact ♦ to Editor the Letters number of genome-wide association studies. 6with schizophrenia supported has been by a histocompatibility complex (xMHC) region on 9.78 ×10 in schizophrenia with prominent fold changes (P 11genes1B, were significantly expression the all decreased of expressed step inthis (Figure 1A).As shown inFigure for multiple comparisons and thus were regarded as differentially values < reached nominal inStudent significance t genescoding were above limit the of detection. Sixty genes sample by set 384-well plate TaqMan real-time PCR. Third, we evaluated result the independent inthe validation previous positive genes by 384-well plate TaqMan PCR. real-time reprepared RNA samples and set reassayed discovery inthe of located xMHC inthe region were investigated, with expression samplediscovery Expressions set. of 162protein-coding genes Fisher to screen differential Scientific) expressions inthe PCR–basedtime TaqMan low-density arrays (TLDAs) (Thermo intotranscribed complementary DNA. First, we applied real- were isolated,Leukocytes and RNA and was extracted reverse morning inthe collected after overnight fasting (10–12 hours). and institutional board review approval. samples Blood were patients and controls were recruited after informed consent and Statistical Manual of Mental Disorders ( schizophrenia according Diagnostic the to Fourth the Edition of patients were first-episode, drug-naïve, and with diagnosed (41patientsset and 37controls) (Supplementary eTable 1).All (24 patients and 24healthy controls) and 1validation sample to schizophrenia identify candidate genes. expressions xMHC inthe region leukocytes inperipheral blood platforms and 2sample to investigate sets -coding gene we utilize 2real-time polymerase chain reaction (PCR)–based provide valuable candidate genes for future validation. Here scrutinization of xMHC inthe region can to attributedifficult association genes. the Atargeted to specific genedense clusters, and strong linkage disequilibrium, it is xMHCthe region is featured by numerous polymorphisms, identified asidentified candidate genes (P among 9genes which with statistical were significance finally exhibited trend decreased of expressions inschizophrenia, independentthe validation sample 11 genes the set, consistently would gene affect expressions inschizophrenia. HIST1H4I Genes future investigation to determineand whether how genes these Zinc-binding usually regulate transcriptions. It deserves zinc-binding proteins, ie, [after adjustment for smoking status], fold changes: 0.61to 0.83). ( supportedreal-time which PCRmethod, evengreater changes was with successfully 384-well confirmed the plate TaqMan Reveals 9Candidate inSchizophrenia Genes the xMHCRegion ExpressionGene Profiling of P values: 3.32 ×10 values: ACTB as an internal beta) (actin, standard. Second, we Results. Methods. The 2 sample include sets sample 1discovery set In 9validated the genes,* To the Editor: .05), while 11 of them survived Bonferroni survived correction them 11of .05), while −6 to 3.74 ×10 to In the TLDA experiment, 127 of the 162protein- In TLDA the the experiment, 127of −7 Strong extended majorthe association of to 2.29 ×10 to −4 , fold changes: 0.61to 0.77).This result ZNF184, ZSCAN12,and ZNRD1. there were 3genes that encode values: 5.27 ×10 values: −3 , fold changes: 0.45to 0.76).In tests (unadjusted P 1 However, since −11 DSM-IV). All to 3.58 ×10 to values: −3 −3

immune cells. systemnervous are concomitant blood with altered of function main neurotransmitter and hormonal systems central inthe to blood-brain or barrier tissue specificity, disturbances inthe limited power changes to directly reflect inbrain tissues due schizophrenia. Although changes have inperipheral blood genes implies malfunctioning of inflammation processes in was observed within and aroundwas observed genes inschizophrenia. and 6. 5. 4. 3. 2. 1. R * subnetwork. schizophrenia and as identified ahub gene inagene expressional of synthesis and promotes inflammation. expression Decreased in adaptive immune response. enhances ABCF1 protein immune-related genes. BTN3A3 is involved inT-cell activity patients and siblings. their structure found and been has function schizophrenia also in both Abnormal expression of genes related to nucleosome and histone is suggested. for schizophrenia. Future validation inother larger populations candidatethe genes as we genetic may identified markers serve controlled sample background and consistency the in2subsets, Although our sample sizesare relatively given small, strictly the expressionsthe of genes certain may have therapeutic effects. immune cells in schizophrenia. Compounds that can normalize xMHCin the region and highlights potential malfunctioning of This resultsets. suggests transcriptional a decreased activation PCR–based platforms experimental and 2independent sample schizophrenia patients, which was consolidated by 2 real-time xMHC-region gene expressions and infirst-episode drug-naïve psychiatric symptoms. might normal affect inflammation process inbrains and cause suggestswhich gene expression changes leukocytes, in blood lymphaticthe vessel systemof central inthe system, nervous study inschizophrenia patients. expression of reported inaprevious TUBBhas been microarray tubulin that is major the constituent of microtubules. Altered that helps inmitochondrial protein synthesis. TUBBencodes Definitions for the genes appear in the Figure 1 footnote. 1 Figure the in appear genes the for Definitions eferences

had been found had been ofABCF1 whole inthe blood patients with In conclusion, study this down-regulation revealed of 9 2014;75(4):276–283. Psychiatry Biol psychosis. in complex histocompatibility major the at findings studies: association DW. Genome-wide Morris A, Corvin Neuropsychopharmacol Biol Psychiatry Prog disorders. psychiatric potential studying for probe: aneural as HF, Lymphocytes J. Korf Kauffman A, Gladkevich 2012;7(6):e39498. PLoS genes. ONE antipsychotic-use brain-expressed for and enriched of independent is patients schizophrenia of blood whole in network MP, co-expression TF, Boks S, Agene Fuller al. et Jong de Eur J Hum Genet genes. candidate highlights schizophrenia additional meta-analysis GWAS from loci top of analysis QTL Expression (GWAS) Consortium. Schizophrenia PGC DW, al. et Zeegers KR, Eijk van S, Jong de 869–887. Am J Med Genet B Neuropsychiatr Genet relatives. biological and first-degree their with schizophrenia signatures of individuals gene-expression blood in peripheral differences and Similarities al. et N, Nossova WS, Stone SJ, Glatt 753–757. 6p22.1 Nature are with schizophrenia. associated chromosome on variants Common DF, al. et J, Duan Levinson J, Shi HIST1H4C . encode Strong of evidence associations © 2016Copyright Physicians Postgraduate Press, Inc. PubMed doi:10.1002/ajmg.b.31239 PubMed doi:10.1002/ajmg.b.31239 5 Decreased expression Decreased BTN3A3 of and ABCF1 6 Moreover, arecent study doi:10.1371/journal.pone.0039498 PubMed PubMed . 2012;20(9):1004–1008. 3

encodes ribosomal protein ribosomal MRPS18B encodes 4 . 2004;28(3):559–576. . 2004;28(3):559–576.

. 2011;156B(8): ABCF1 7 reported the discovery reported discovery the doi:10.1038/ejhg.2012.38 PubMed and BTN3A3 and are . 2009;460(7256): doi:10.1016/j.pnpbp.2004.01.009 PubMed

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It is illegal to post this copyrighted PDF on any website. e598 For [email protected]. reprints orpermissions, contact ♦ B. Replication and Validation ofDifferentially Expressed Genes Protein-Coding 127Detectable Expressions Gene inthe the A. PerformanceTLDA of Experiment Figure Expression ofGene 1.Results Profiling inthexMHCRegion inSchizophrenia Letters to theEditor d c b a .05. *P <.05. It isIt illegal to post this copyrighted PDF on any website. P values represents value times10to the Genes areGenes mappedintheplotaccording to P values invalidation setare adjustment for after All Pvalues presented here are unadjusted (for negative power, eg, 6.90E-05= in boldface. tests. The finalvalidated 9candidate genesare multiple testing). Pvalues are from Studentt chromosomal location (x-axis) and “–log the TLDA step are annotated by genenames. expressed genesinschizophrenia identified in (P value)” value (y-axis). The 11differentially regarded asstatistically significant. PCR experiments, andonlythesePvalues are in correction TLDA or384-well plate real-time status. smoking ABCF1 Name Gene ATAT1 BRD2 BTN3A3 HIST1H4C HIST1H4I MRPS18B TUBB ZNF184 ZNRD1 ZSCAN12

values that could survive Bonferroni**P values that could survive

0.66 0.61 0.63 0.68 0.72 0.76 0.69 0.70 0.71 0.77 0.72 FC (Discovery Set) (Discovery TLDA 1.49 E-04** 6.90 E-05** 1.85 E-04** 1.84 E-04** 3.74 E-04** 2.76 E-05** 4.74 E-05** 2.13 E-04** 9.78 E-06** 3.19 E-04** 1.71 E-05** P Value c × 10 10 –5 . 384-Well Plate PCR RT 0.69 0.71 0.76 0.63 0.60 0.45 0.54 0.67 0.63 0.66 0.57 FC (Discovery Set) (Discovery fold change, RT PCR =real-time RT change, FC =fold Abbreviations: ABCF1 abbreviations:Gene ATAT1 BTN3A3 2 containing BRD2 =bromodomain HISTIH4C major histocompatibility complex histocompatibility =major HLA HIST1H4I polymerase chainreaction, TLDA array, xMHC -DMA/B C/B I, =class -C/B A I, -A =class class I, F I, -F =class E I, -E =class -DRA/B -DQA/B -DPA/B 1.63 E-04** 3.64 E-04** 2.29 E-03** 4.12 E-04** 5.38 E-07** 3.32 E-07** 8.30 E-07** 3.38 E-05** 3.00 E-04** 1.20 E-04** 1.58 E-05**

= = P Value alpha tubulinacetyltransferase 1 = ATP bindingcassette subfamilyFmember = = = = = butyrophilin subfamily3memberA3 = class II,DPalpha/beta histone cluster 1,H4i class II,DRalpha/beta histone cluster 1,H4c class II,DQalpha/beta class II,DMalpha/beta b

= c extended majorhistocompatibilityextended complex. 384-Well Plate PCR RT 0.81 0.88 0.96 0.81 0.73 0.61 0.70 0.79 0.83 0.74 0.76 FC (Validation Set) (Validation 1.00 E-03** 1.80 E-02* 2.83 E-01 3.58 E-03** 5.02 E-07** 3.05 E-09** 2.00 E-06** 8.00 E-06** 1.41 E-03** 5.27 E-11** 2.58 E-04** = P Value © 2016Copyright Physicians Postgraduate Press, Inc. TaqMan low-density c,d a ZSCAN12 ZNRD1 ZNF184 ZFPs TUBB genes motif-containing TRIMs =tripartite SLC17A1/3 16 serine PRSS16 =protease, genes receptor ORs =olfactory ribosomal MRPS18B =mitochondrial containing 12 member 1/3 protein S18B J ClinPsychiatry 77:5,May 2016 = = zinc fingerprotein genes = tubulin betaclassI = zinc ribbon domaincontainingzinc ribbon 1 = zinc fingerprotein 184 = zinc finger and SCAN domain zinc fingerandSCAN solute carrier family17 solute carrier

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2015;523(7560):337–341. 2015;523(7560):337–341. Nature vessels. lymphatic system nervous central of features functional and TJ, Structural Keyes al. I, et Smirnov A, Louveau Pub Chunling Wan,PhD Supplementary material followsSupplementary thisletter. Jingyan Ding, MS [email protected] Liwen Tan,PhD Chao Yang,PhD Xiaowen Hu, BS Xuhan Yang,BS Liya Sun,PhD Ying Qing, BS Jie Jiang, BS Lin He, PhD . a, c, a,‡ a,‡ * * b a a a a a Supplementary material: Central Hospital, Xiangya University, South Changsha, China. Second the of Institute Health Mental from were Both study. this in processing pre- sample and recruiting participant in contributions their for MM, Peng, Acknowledgment: manuscript. the of approval and review, preparation, or data; the of analysis and management, collection, study; the of conduct and design the in role no sponsor:the areRole organizations institutions public funding and had The of (81271486). China of Foundation (2012CB910100) Science Nature National the and Funding/support: None. interest: of conflicts Potential as corresponding authors. * ‡ Hunan Province Technology Institute of Psychiatry, Changsha, China Province, Hunan of Health Mental and Psychiatry of Laboratory Key University, © Copyright 2016Physicians Postgraduate Press, Inc. dx.doi.org/10.4088/JCP.15l10156 2016;77(5):e597–e599 J ClinPsychiatry c Mental Health Institute of the Second Xiangya Hospital, Central South South Central Hospital, Xiangya Second the of Institute Health Mental Dr Sun and Ms Qing contributed equally to this letter. this to equally contributed Qing Ms and Sun Dr Dr Tan and Dr Wan contributed equally to this letter, and both qualify to serve serve to qualify both and letter, this to equally Tan Wan contributed Dr Dr and © 2016Copyright Physicians Postgraduate Press, Inc. This work was supported by the 973 Program 973 Program the by supported was work This The authors thank Jinghong Chen, MM, and Xuemei Xuemei and MM, Chen, Jinghong thank authors The See accompanying pages. pages. accompanying See Letters to theEditor

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It is illegal to post this copyrighted PDF on any website. Supplementary Material

Article Title: Gene Expression Profiling of the xMHC Region Reveals 9 Candidate Genes in Schizophrenia

Author(s): Liya Sun, PhD; Ying Qing, BS; Xuhan Yang, BS; Chao Yang, PhD; Jie Jiang, BS; Xiaowen Hu, BS; Jingyan Ding, MS; Lin He, PhD; Liwen Tan, PhD; Chunling Wan, PhD

DOI Number: dx.doi.org/10.4088/JCP.15l10156

List of Supplementary Material for the article

1. eTable 1 Demographic data of the sample sets

Disclaimer This Supplementary Material has been provided by the author(s) as an enhancement to the published article. It has been approved by peer review; however, it has undergone neither editing nor formatting by in-house editorial staff. The material is presented in the manner supplied by the author.

© Copyright 2016 Physicians Postgraduate Press, Inc. It is illegal to post this copyrighted PDF on any website. ♦ © 2016 Copyright Physicians Postgraduate Press, Inc. Supplementary eTable 1. Demographic data of the sample sets

Sample set 1 (discovery set) Sample set 2 (validation set)

Normal Items Patients Normal Controls Patients Controls (n=24) (n=24) (n=41) (n=37)

Male / Female 12 / 12 12 / 12 15 / 26 19 / 18

Age (year, mean ± 25.17 ± 0.70 24.71 ± 0.34 27.59 ± 1.52 29.03 ± 1.06 SEM)

Height (cm, mean ± 164.00 ± 1.41 163.79 ± 2.01 162.27 ± 1.22 165.78 ± 1.15 SEM)

Weight (kg, mean ± 56.75 ± 2.17 56.94 ± 1.81 58.61 ± 2.02 64.2 ± 2.54 SEM)

Waist

circumference(cm, 77.38 ± 1.75 75.92 ± 1.73 78.07 ± 1.67 79.19 ± 1.31

mean ± SEM)

BMI (kg/m2, mean ± 21.02 ± 0.64 21.12 ± 0.35 22.13 ± 0.61 23.23 ± 0.82 SEM)

Nonsmoker/Smoker 24 / 0 24 / 0 35 / 6 23 / 14

Location 7 / 2 /15 6 / 1 / 17 8 / 4 / 29 21 / 4 / 12 (city/village/rural)

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