A Point of Rarity in Genetic Risk for Bipolar Disorder and Schizophrenia

WEB-ONLY CONTENT Rare Copy Number Variants A Point of Rarity in Genetic Risk for Bipolar Disorder and Schizophrenia

Detelina Grozeva, MSc; George Kirov, PhD, MRCPsych; Dobril Ivanov, MSc; Ian R. Jones, PhD, MRCPsych; Lisa Jones, PhD; Elaine K. Green, PhD; David M. St Clair, MD, PhD; Allan H. Young, PhD, FRCPsych; Nicol Ferrier, PhD, FRCPsych; Anne E. Farmer, PhD, FRCPsych; Peter McGuffin, PhD, FRCPsych; Peter A. Holmans, PhD*; Michael J. Owen, PhD, FRCPsych*; Michael C. O’Donovan, PhD, FRCPsych*; Nick Craddock, PhD, FRCPsych*; for the Wellcome Trust Case Control Consortium

Arch Gen Psychiatry. 2010;67(4):318-327

14 RESULTS schizophrenia article. In cases, this is caused by the ex- clusion of schizoaffective cases in the current analysis. The differences with regard to controls are caused by re- COMPARISON OF BURDEN OF COPY NUMBER analysis of a small subset of them. Some of the control VARIANTS ACCORDING TO SIZE BETWEEN data were processed with the use of different reference BIPOLAR CASES, CONTROLS, AND batches, which enabled us to include more controls in SCHIZOPHRENIA CASES the current analysis. We compared our bipolar disorder cases against our set of schizophrenia cases from the same population who had CNVs THAT OCCURRED MORE OFTEN IN been examined by the same methods (n=440).14 The copy BIPOLAR DISORDER CASES THAN CONTROLS number variants (CNVs) were classified into size cat- egories as in our previous report. eTable 1 shows the Although we did not observe an overall increase of CNV results. Compared with bipolar disorder, in the schizo- burden in bipolar cases compared with controls, some phrenia sample we observed a significant excess of large individual CNVs were more common in cases than con- deletions (PϽ.001) and total large CNVs (PϽ.001) and trols. None was significantly associated after correction a trend for excess large duplications (P=.053). for multiple testing. Those CNVs for which there was an uncorrected nominal significance level of ␣Յ.05 are pre- DIFFERENCES BETWEEN SAMPLES USED sented in eTable 2. IN SCHIZOPHRENIA ANALYSIS REPORTED PREVIOUSLY AND SCHIZOPHRENIA-CONTROL ANALYSIS OF CNVs IN REGIONS PREVIOUSLY COMPARISONS IN THIS ANALYSIS REPORTED TO SHOW ASSOCIATIONS IN BIPOLAR DISORDER SAMPLES There are small differences with respect to the analyzed individuals reported herein and the ones described in our We investigated whether CNV regions previously hypoth- esized to be involved in susceptibility to bipolar disorder *Indicates research team members who played a major role in were affected by CNVs in our data. The CNVs on chro- supervising and coordinating the bipolar research described mosomes 1p34.3, 14q23.3, and 22q12.3, affecting GLUR7 herein. (official symbol, GRIK3), AKAP5, and CACNG2, respec-

eTable 1. Comparisons of Rates of CNVs Between Controls and BD and SZ Casesa

Size CNVs per BD to Control Ratio SZ to Control Ratio BD to SZ Ratio CNV Type Range, kb Control (P Value) (P Value) (P Value) Deletion 100-200 0.104 0.801 (.03) 0.771 1.045 200-500 0.102 0.864 1.141 0.758 500-1000 0.012 1.264 0.938 1.348 Ͼ1000 0.007 0.579 3.189 (.004) 0.181 (Ͻ.001) Total 0.225 0.85 (.01) 1.032 0.825 Duplication 100-200 0.093 0.982 0.882 1.126 200-500 0.155 1.051 0.951 1.105 500-1000 0.050 0.868 1.147 0.757 Ͼ1000 0.023 0.839 1.570 0.53 (.053) Total 0.321 0.990 1.007 0.984 Total (deletionϩduplication) 0.546 0.930 1.017 0.918

Abbreviations: BD, bipolar disorder; CNV, copy number variant; kb, kilobase; SZ, schizophrenia. a Significant differences are in boldface type. P values are not corrected for multiple testing. Those that would survive correction are the excess of large (Ͼ1 megabase) deletions in SZ compared with either controls or BD cases.

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 eTable 2. Regions Showing More CNVs in Cases Than Controls at a Nominal Significance of PՅ.05

BD P Value, Locusa Start bpb End bpb Type Casesc Controlsc Fisher Exact Test Gene 1q25.1 173 769 777 173 978 862 dup 5 1 .03 TNR 9q31.1 104 826 097 104 885 068 del/dup 3 0 .05 None 12p11.21 31 202 250 31 301 551 dup 19 16 .03 OVOS2 18p11.21-11.1 14 694 694 15 092 421 dup 3 0 .05 ANKRD30B 19p12 20 001 614 20 177 979 del/dup 5 1 .03 ZNF682, ZNF90, ZNF486 19p12 24 013 968 24 295 825 dup 3 0 .05 ZNF254

Abbreviations: BD, bipolar disorder; bp, base pair; CNV, copy number variant; del, deletion; dup, duplication. a Locus refers to the chromosome band. b Start bp and end bp provide the start base pair position and the end base pair position of the CNVs at the specified locus. c Numbers of individuals with BD and controls harboring the CNV.

eTable 3. Percentage of Samples Carrying at Least 1 Singleton CNV in Our Study Compared With the Study of Zhang et al20

Current Study, No. (%) Study of Zhang et al, No. (%)

Single CNV Type Cases With CNV Controls With CNV Cases With CNV Controls With CNV Deletion 101 (6.0) 173 (6.2) 162 (16.2) 127 (12.3) Duplication 121 (7.1) 234 (8.3) 197 (19.7) 197 (19.1) Deletionϩduplication 187 (11.0) 329 (11.7) 320 (32.0) 299 (29.0)

Abbreviation: CNV, copy number variant.

tively, were studied.21 No deletions or duplications were not in any control. Where several CNVs mapped to the found. The other studied locus was 3q13.3, reported to same region, they did not always fully overlap. The start harbor duplication in the GSK3B gene.22 This gene codes and end points in the table refer to the total region cov- for glycogen synthase kinase, a key player in the Wnt sig- ered by such a cluster of CNVs. The list contains several naling pathway and a target of lithium.22 No CNVs were intriguing candidate genes (eg, neuroligin 1, neuregu- detected at this locus in bipolar cases. lin 3, and the ␣-2 catenin), although we have no spe- cific evidence that any contribute to susceptibility to bi- SINGLETON EVENTS: COMPARISON polar disorder. OF OUR DATA WITH PREVIOUS STUDIES ANALYSIS OF CNVs THAT DISRUPT GENES With the use of PLINK,24 deletions-only and duplications- only data sets were generated. In these data sets, a dele- The global burden of CNVs in cases and controls with tion was still defined as a single-occurrence event even respect to genes that are deleted, duplicated, or dis- if there was duplication at the same region. The same rule rupted by CNVs was estimated by using PLINK24 1.05. was applied to duplications. Therefore, the number of Only CNVs that overlapped with at least 1 gene, on single-occurrence deletions and single-occurrence du- the basis of hg18 genomic coordinates, were taken into plications does not sum to the total number of single- account. This analysis was performed for all CNVs and occurrence events. then for the CNVs that occurred only once in the data. The power in our sample at ␣=.05 to detect a significant difference with the effect size observed by Zhang et CNVs PREVIOUSLY ASSOCIATED al20 (␦=0.11) was 95%. For comparison, the power to de- WITH SCHIZOPHRENIA tect a significant difference (␦=0.11) with their sample size was 71% (␣=.05). As mentioned in the “Comment” Table 4 in the main article shows the main chromo- section, the Zhang et al study20 used a higher-resolution somal regions with CNVs associated with schizophre- array (Affymetrix 6.0) and had a smaller sample size. Both nia and the number of the corresponding CNVs in the of these factors can lead to the observation of a higher bipolar disorder cases and controls. Herein we provide number of singleton CNVs in their study and could ex- more information on these CNVs. plain the differences from our study (eTable 3). A deletion at 15q13.3 loci has been found to be associated with schizophrenia in 2 large studies of schizo- ANALYSIS OF GENES WITHIN CNVs phrenia.12,13 Deletion in the same region was reported to FOUND ONLY IN CASES be associated with mental retardation and seizures.16 No deletions were observed in bipolar disorder, but 2 cases We provide a list of genes within all CNVs that were pres- and no controls were found to harbor duplications in this ent in cases but not in controls (eTable 4). The list in- region (Fisher exact test, P=.14). The significance (or oth- cludes not only singleton CNVs (those found once in the erwise) of duplications in this region is not yet known data set) but also CNVs found in more than 1 case, but from other studies.

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No. of Chr Start bp End bp Type NCBI Genes Observations 1 45 592 076 45 881 891 3 TESK2, LOC126661, MMACHC, PRDX1, AKR1A1, NASP, CCDC17, 1 GPBP1L1 1 46 131 935 46 268 021 3 MAST2 1 1 65 071 676 65 413 912 3 JAK1, MIRN101-1, AK3L1, AK3L2 1 1 86 324 452 86 589 985 1 COL24A1, ODF2L 1 1 88 847 985 89 032 679 3 PKN2 1 1 107 551 303 107 823 281 3 NTNG1 1 1 143 576 984 143 916 898 1 PDE4DIP, FLJ21272, SEC22B 1 1 152 936 201 153 149 168 1 KCNN3 1 1 166 729 757 167 024 066 1 XCL2, XCL1, DPT 1 1 173 980 586 174 202 365 3 LOC100128153, RFWD2 1 1 217 365 162 217 613 673 1 LOC643723, LYPLAL1 1 1 221 702 789 222 127 692 3 LOC644151, LOC653428, CAPN8, CAPN2, TP53BP2 1 1 231 361 905 231 514 154 1 PCNXL2 1 1 244 896 239 245 307 486 3 C1orf71, SCCPDH, LOC100130097, LOC149134, AHCTF1, ZNF695, 1 ZNF670 2 155 674 306 842 3 LOC727818, SH3YL1, ACP1, FAM150B 1 2 9 950 762 10 157 626 3 TAF1B, GRHL1, UNQ5830, KLF11, LOC100131506, CYS1 1 2 46 875 500 47 239 348 3 LOC388948, MCFD2, TTC7A, LOC100129286, C2orf61 1 2 49 008 203 49 120 697 1 FSHR 1 2 49 149 900 49 602 914 1 FSHR 1 2 53 738 562 53 909 774 3 ASB3, CHAC2, C2orf30 1 2 67 026 445 67 574 931 1 LOC644838, ETAA1 1 2 80 501 388 81 016 718 1 CTNNA2 1 2 105 308 220 105 583 762 1 TGFBRAP1, C2orf49, FHL2, LOC728966 1 2 108 755 278 109 814 503 1 RANBP2, CCDC138, EDAR, SH3MD4, LOC100132457, LOC729164, 1 SEPT10, ANKRD57, LOC100131577 2 137 213 276 137 516 286 1 THSD7B 1 2 175 063 486 175 545 089 1 LOC100130325, WIPF1, LOC100133109, CHRNA1, CHN1 1 2 211 762 892 212 453 633 3 ERBB4 1 2 230 634 380 230 823 051 1 SLC16A14, SP110, SP140 1 3 96 226 577 858 1 LOC642891, CHL1 1 3 6 731 867 7 502 372 3 GRM7 2 3 56 538 380 56 738 659 3 CCDC66, C3orf63, ARHGEF3 1 3 65 782 031 66 651 118 3 MAGI1, SLC25A26, LRIG1 1 3 108 045 770 108 523 998 1 LOC344595 1 3 109 343 496 109 735 186 3 IFT57, HHLA2, MYH15 1 3 126 612 179 126 918 638 3 SNX4, OSBPL11 1 3 159 972 476 160 172 525 1 MFSD1 1 3 169 645 563 170 273 620 3 LOC389174, MIRN551B, LOC253820, C3orf50 1 3 175 127 179 175 441 056 3 NLGN1 1 3 191 379 157 191 640 477 1 CLDN1, CLDN16, UNQ846 1 4 5 108 518 5 215 976 1 STK32B 1 4 74 735 392 74 864 580 3 IL8 1 4 78 951 173 79 103 165 3 CNOT6L, MRPL1 1 4 95 781 900 96 255 099 3 PDLIM5, LOC728442, BMPR1B 1 4 100 189 816 100 342 799 1 METAP1, ADH5, ADH4 1 4 113 529 096 113 893 894 3 ALPK1, NEUROG2, LOC91431, C4orf21, LARP7, MIRN367, MIRN302D, 1 MIRN302A, MIRN302C, MIRN302B 4 114 884 938 115 470 791 3 CAMK2D, ARSJ 1 4 120 432 331 120 685 299 3 USP53, LOC401152, FABP2, LOC729249, FLJ14186, LOC645513, PDE5A 1 4 144 067 928 144 176 340 1 LOC729675 1 4 154 588 412 155 163 845 3 KIAA0922, TLR2, RNF175, SFRP2 1 4 162 419 238 162 568 359 1 FSTL5 1 4 177 932 828 178 065 111 3 VEGFC 1 4 178 467 820 178 584 108 3 NEIL3 1 4 184 350 807 184 681 169 3 WWC2, CLDN22, LOC100132463, LOC100131811, CDKN2AIP, ING2 1 5 1 695 155 1 883 374 3 MRPL36, NDUFS6 1 5 2 409 789 2 732 511 3 LOC100133292 1 5 19 203 456 19 757 247 3 CDH18 1 5 20 009 671 20 700 523 3 CDH18 1 5 22 346 817 23 379 949 3 CDH12 1 5 37 541 744 37 660 208 3 WDR70 1 5 58 867 115 59 095 265 1 PDE4D, MIRN582 1 5 60 152 290 60 767 215 3 ELOVL7, ERCC8, NDUFA12L, DKFZP686E2158, ZSWIM6, LOC728153 1

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No. of Chr Start bp End bp Type NCBI Genes Observations 5 95 457 099 95 693 430 1 LOC441097 1 5 99 678 948 100 293 602 3 LOC100133050, LOC100132020, TMEM157, ST8SIA4 1 5 110 682 996 110 894 149 1 CAMK4, STARD4 1 5 126 925 062 127 404 930 1 CTXN3, LOC728586 1 5 130 387 114 130 720 739 3 HINT1, LYRM7, CDC42SE2 1 5 180 460 061 180 624 927 3 TRNAL-AAG, TRNAV9, OR2V2, TRNAV-AAC, TRNAV-CAC, 1 TRNAP-UGG, TRNAT1, TRIM7, TRNAA-UGC, TRNAK-CUU, TRIM41, GNB2L1, SNORD96A, SNORD95, TRIM52 6 119 769 496 352 3 FLJ43763, DUSP22, IRF4, EXOC2, LOC727827, LOC642335 1 6 63 009 959 63 255 396 3 KHDRBS2 1 6 68 334 871 68 703 877 1 LOC100128757 1 6 74 168 491 74 593 230 3 DDX43, C6orf150, MTO1, EEF1A1, SLC17A5, CD109 1 6 91 481 484 94 497 325 3 EPHA7 1 6 106 604 408 106 937 568 3 PRDM1, ATG5 2 6 125 557 330 125 906 207 3 TPD52L1, HDDC2 1 6 127 357 460 127 691 043 1 RSPO3, RNF146, ECHDC1 1 7 12 695 794 13 112 747 3 ARL4A 1 7 16 058 262 16 586 160 3 LOC729920, SOSTDC1, LOC100129771, LOC100129335 1 7 49 540 495 49 894 866 3 VWC2, LOC100128734 1 7 63 200 647 63 487 624 1 LOC730291, ZNF679, LOC728927 1 7 141 274 408 141 407 299 1 CLEC5A, TAS2R38, MGAM 1 7 145 582 575 145 743 361 1 CNTNAP2 1 7 157 369 323 157 513 462 1 PTPRN2 1 8 1 987 919 2 374 301 1 MYOM2 1 8 18 566 441 19 801 599 3 PSD3, LOC100131275, LOC100128993, SH2D4A, ChGn, 2 LOC100130604, INTS10 8 82 552 799 82 735 204 3 FABP4, LOC646486, LOC100129523, IMPA1 1 8 86 461 282 86 722 738 3 CA1, CA3, LOC100132709, CA2 1 8 87 220 440 87 426 294 3 ATP6V0D2, LOC100128962, SLC7A13, WWP1 1 8 124 096 689 126 212 968 1 DERL1, WDR67, TRNAM-CAU, FAM83A, LOC100131726, C8orf76, 1 ZHX1, ATAD2, MIRN548D1, C8orf32, FBXO32, C8ORFK36, ANXA13, FAM91A1, FER1L6, TMEM65, TRMT12, RNF139, TATDN1, NDUFB9, MTSS1, ZNF572, SQLE, KIAA0196, NSMCE2 8 138 893 819 139 296 115 3 FLJ45872, FAM135B 1 8 145 891 814 146 125 907 3 ZNF251, ZNF34, RPL8, ZNF517, LOC100130027, LOC100129596, 1 ZNF7, COMMD5, ZNF250 9 9 423 671 9 550 338 1 PTPRD 1 9 18 416 823 18 776 181 1 ADAMTSL1 1 9 70 797 218 71 143 539 3 PIP5K1B, PRKACG, FXN, LOC100131414, TJP2, C9orf61 2 9 71 528 430 72 053 813 3 PTAR1, C9orf135, MAMDC2 1 9 104 723 219 104 885 068 1 CYLC2 1 9 118 549 097 118 655 895 1 ASTN2 1 9 131 966 512 132 099 642 3 FREQ 1 10 1 412 160 6 132 099 3 ADARB2, LOC642384, C10orf109, LOC100129465, LOC728209, 1 LOC727878, PFKP, LOC100133296, PITRM1, KLF6, LOC100130652, LOC727894, LOC728544, LOC100128356, LOC338588, AKR1CL2, tAKR, AKR1C1, AKR1C2, AKR1C3, AKR1CL1, AKR1C4, UCN3, TUBAL3, NET1, CALML5, CALML3, ASB13, C10orf18, GDI2, TRNAV-UAC, ANKRD16, FBXO18, IL15RA, IL2RA 10 29 684 578 29 911 570 3 SVIL, MIRN604 1 10 41 956 473 43 152 050 3 LOC100131936, LOC653097, LOC100132349, CCNYL2, MGC16291, 1 ZNF37B, LOC100128934, ZNF33B, LOC100129622, LOC283028, BMS1, RET, GALNACT-2, RASGEF1A 10 47 063 957 51 229 942 3 LOC728684, ANXA8L2, LOC728449, FAM21B, ASAH2C, LOC727950, 1 LOC100132209, CTGLF6, LOC100133093, ANXA8, LOC653110, ZNF488, RBP3, GDF2, GDF10, PTPN20B, FRMPD2L1, LOC644021, LOC644054, LOC100133265, FRMPD2, MAPK8, ARHGAP22, C10orf64, LOC642343, WDFY4, LRRC18, C10orf72, C10orf73, LOC728883, C10orf128, LOC100132730, C10orf71, LOC100130757, DRGX, LOC100128032, ERCC6, PGBD3, CHAT, SLC18A3, C10orf53, OGDHL, LOC727726, FAM21D, LOC728955, CTGLF5, TIMM23B, LOC100133089, CTGLF4, MSMB 10 51 462 803 51 815 423 3 FAM21A, ASAH2, SGMS1 1 10 57 761 600 58 753 290 1 ZWINT 2 10 65 228 767 68 139 445 3 CTNNA3 2

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No. of Chr Start bp End bp Type NCBI Genes Observations 10 81 567 623 81 966 905 3 LOC642361, FAM22E, SFTPD, LOC642521, LOC642538, C10orf57, 1 PLAC9, ANXA11 10 83 980 248 84 561 902 1 NRG3 1 10 93 824 133 94 106 871 1 CPEB3, LOC100130772, MARCH5 1 10 98 850 750 99 028 923 3 SLIT1, ARHGAP19 1 10 112 373 593 112 648 954 3 RBM20, LOC282997, LOC100132573, PDCD4, LOC92482 1 11 201 447 373 554 1 RIC8A, SIRT3, PSMD13, NLRP6, ATHL1, IFITM5, IFITM2, IFITM1, IFITM3, 1 B4GALNT4 11 201 447 430 343 3 RIC8A, SIRT3, PSMD13, NLRP6, ATHL1, IFITM5, IFITM2, IFITM1, IFITM3, 1 B4GALNT4, PKP3, SIGIRR, TMEM16J 11 4 120 756 4 232 709 3 LOC196120, LOC390031 1 11 54 596 906 55 739 642 3 TRIM48, OR4A16, OR4A15, OR4C15, OR4C16, OR4C11, OR4P4, OR4S2, 2 OR4C6, OR5D13, OR5D14, OR5L1, OR5D18, OR5L2, OR5D16, SPRYD5, OR5W2, OR5I1, OR10AG1, OR5F1, OR5AS1, OR8I2, OR8H2, OR8H3, OR8J3, OR8K5, OR5J2 11 54 937 013 55 112 298 1 OR4C15, OR4C16 1 11 68 304 836 68 542 057 3 CPT1A, MRPL21, IGHMBP2, MRGPRD, MRGPRF 1 11 69 837 338 70 074 717 3 PPFIA1, CTTN, SHANK2 1 11 121 963 463 122 151 248 3 UBASH3B 1 11 128 156 738 128 275 086 3 FLI1, KCNJ1, KCNJ5, C11orf45 1 12 971 525 1 119 511 3 LOC100130219, ERC1 1 12 2 022 432 2 541 278 3 CACNA1C 1 12 5 094 169 5 260 067 1 LOC387826 1 12 24 357 971 24 519 700 3 SOX5 1 12 38 872 898 39 028 521 1 LRRK2 1 12 52 503 589 52 628 593 1 HOXC13 1 12 71 297 905 71 840 265 3 TRHDE 1 12 77 701 054 77 869 712 1 LOC100129021 1 12 86 803 928 87 584 968 1 C12orf50, C12orf29, CEP290, TMTC3, KITLG 1 12 95 329 061 95 526 343 1 C12orf55 1 12 97 551 176 97 756 322 3 IKIP, APAF1, ANKS1B 1 12 100 749 452 101 055 184 1 DRAM, CCDC53, NUP37, C12orf48 1 12 121 217 208 121 868 133 3 LRRC43, IL31, B3GNT4, DIABLO, VPS33A, CLIP1, TRNAD-GUC, ZCCHC8, 1 RSRC2, KNTC1, GPR109A, GPR109B, GPR81, DENR, CCDC62 12 127 358 570 127 647 495 3 TMEM132C 1 13 30 803 641 30 977 080 3 B3GALTL 1 13 31 177 504 31 439 521 3 RXFP2 1 13 48 259 408 48 519 957 1 LOC647131, FNDC3A 1 13 82 905 273 83 506 735 3 SLITRK1 1 14 21 604 337 21 840 943 3 TRA@, TRAV22, TRAV23DV6, TRDV1, TRAV24, TRAV25, TRAV26-1, 4 TRAV8-7, TRAV27, TRAV29DV5, TRAV30, TRAV26-2, TRAV34, TRAV35, TRAV36DV7, TRAV38-1, TRAV38-2DV8 14 33 465 639 33 586 331 3 EGLN3 1 14 50 241 969 50 382 223 1 NIN 1 14 52 385 742 52 525 010 3 FERMT2 1 15 58 226 926 58 465 642 3 ANXA2 1 15 74 654 705 74 895 929 1 SCAPER 1 15 82 306 210 82 555 245 3 ADAMTSL3 1 15 99 212 626 100 060 877 1 LOC145757, ALDH1A3, LRRK1, CHSY1, SELS, SNRPA1, PCSK6, TM2D3, 1 TARSL2 16 5 059 873 5 217 562 1 ALG1, FAM86A 1 16 6 490 401 6 854 359 3 A2BP1, LOC100131413 1 16 7 134 152 7 356 599 3 A2BP1 1 16 8 760 561 8 866 827 1 ABAT, TMEM186, PMM2, LOC100132944, LOC100130283, CARHSP1, 1 LOC100129895 16 25 870 606 26 088 563 3 HS3ST4 1 16 26 422 573 27 239 475 3 C16orf82, JMJD5, NSMCE1, IL4R 1 16 45 086 927 45 948 101 3 FLJ43980, LOC100128802, SHCBP1, VPS35, ORC6L, MLCK, LOC388272, 1 GPT2, DNAJA2, NETO2, LOC100127930, ITFG1 16 76 340 176 76 766 570 1 KIAA1576, CLEC3A, WWOX 1 16 76 901 619 77 102 849 1 WWOX, LOC645947 1 16 87 678 937 87 804 936 1 ACSF3, C16orf81, LOC400558, CDH15, LOC146429 1 17 9 975 140 10 275 516 3 GAS7, LOC100129677, MYH13, LOC100128560, MYH8 1 17 14 093 529 15 357 533 3 HS3ST3B1, LOC100131109, LOC388339, FLJ45831, PMP22, TEKT3, 1 CDRT4, FAM18B2

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No. of Chr Start bp End bp Type NCBI Genes Observations 17 16 659 493 17 062 349 3 LOC100129981, LOC96597, LOC100129535, TNFRSF13B, 1 LOC100128283, M-RIP, LOC201164, FLCN 17 31 923 810 33 308 219 3 ZNHIT3, MYO19, PIGW, GGNBP2, MGC4172, MRM1, LOC727862, LHX1, 1 AATF, ACACA, C17orf78, TADA2L, DUSP14, AP1GBP1, LOC100131822, DDX52, HNF1B, LOC284100 17 78 476 537 78 599 918 3 TBCD, B3GNTL1 1 18 12 246 238 12 506 082 3 CIDEA, TUBB6, AFG3L2, SLMO1, SPIRE1 1 18 14 597 916 15 092 421 3 ANKRD30B, LOC647983, LOC100131500 3 18 28 682 047 29 322 188 3 C18orf34 1 18 45 714 083 45 940 515 1 MYO5B 1 19 7 035 307 7 181 438 1 ZNF557, LOC100131165, INSR, LOC100128567 1 19 19 969 224 20 657 868 3 ZNF682, ZNF90, LOC729903, ZNF486, FLJ44894, ZNF626 5 19 23 605 608 24 295 825 3 ZNF675, ZNF681, LOC730087, ZNF254 3 19 44 819 692 45 042 569 1 LOC148003, LOC400696, LGALS14, CLC, DYRK1B, FBL 1 19 59 886 999 60 014 905 1 KIR3DL3, KIR2DL3, KIR2DL1, KIR2DL4 1 20 116 466 352 407 1 DEFB128, DEFB129, DEFB32, C20orf96, ZCCHC3, SOX12, NRSN2, 1 TRIB3, RBCK1 20 25 794 494 26 236 535 3 C20orf91, LOC284801, MIRN663 1 22 24 873 287 25 340 497 3 SEZ6L, FLJ38343, ASPHD2, LOC100128401, HPS4, SRRD, TFIP11, 1 TPST2, LOC100130561, CRYBB1 23 142 664 2 770 060 1 PLCXD1, GTPBP6, PPP2R3B, SHOX, LOC442442, CRLF2, CSF2RA, 5 IL3RA, SLC25A6, LOC729629, CXYorf2, ASMTL, P2RY8, SFRS17A, ASMT, DHRSX, ZBED1, LOC100130595, LOC401577, CD99, XG, GYG2 23 22 008 574 22 149 641 3 PHEX 1 23 35 570 709 35 917 535 3 MAGEB16, CXorf22 1 23 37 739 464 37 857 804 3 SYTL5 1 23 56 824 308 57 065 815 3 SPIN3 1 23 86 509 912 86 714 253 3 KLHL4 1 23 139 671 186 139 903 522 3 CDR1 1 23 143 459 775 146 067 963 1 SPANXN1, SLITRK2, LOC100129095, CXorf1, MIRN890, MIRN888, 1 MIRN892A, MIRN892B, MIRN891B, MIRN891A, LOC100128265, LOC100133053, LOC100129239, LOC100132556 23 144 046 112 144 430 381 3 SPANXN1 1 23 151 732 760 151 922 021 3 CETN2, NSDHL, ZNF185, PNMA5 1

Abbreviations: bp, base pair; Chr, chromosome; CNV, copy number variant; NCBI, National Center for Biotechnology Information.

A region on 16p11.2 has been implicated in schizo- proximately 1.5-Mb region. No duplications or dele- phrenia11 and autism.10 In our sample, duplications at tions were observed in the controls. 16p11.2 were found in 3 cases and 1 control, a 5-fold in- Although deletions in the velocardiofacial syndrome crease in frequency (0.2% vs 0.04%; Fisher exact test, region have been implicated in bipolar disorder,27 we did P=.15). The deletion associated with this region was found not observe any deletions in the bipolar data set. We found in none of the cases and in 3 controls. It should be noted 8 people from the control data set harboring a recipro- that the 16p11.2 locus is covered by 7 markers on the NspI cal duplication in this region (6 of them spanning the full array. Therefore, it would have been filtered out by the qual- region). All were part of the UK Blood Service sample set. ity control criterion of 10 or more consecutive markers It has been suggested that the microduplication of 22q11.2 on each array. To target this region, data for every indi- shares some characteristics with velocardiofacial syn- vidual who showed a CNV in this region, detected by the drome.28-30 Our data add strong evidence against the hy- single-nucleotide polymorphisms (SNPs) on the StyI ar- pothesis that the duplication of 22q11.2 predisposes to ray, were individually inspected with the Affymetrix Geno- bipolar disorder or schizophrenia. typing console v2.1 software. This CNV was accepted if, in addition to the SNPs on the StyI array, all markers on ANALYSIS OF AFFECTED the NspI array within this region also showed a deviation FAMILY MEMBERS in the log2 ratios in the same direction. A deletion on the short arm of chromosome 17 (17p12) In 2 of the individuals with CNVs included in Table 4 in has been found to be associated with schizophrenia the main article, we had DNA from both parents, and the (PϽ.001).14 PMP22, one of the affected genes in this re- father was also affected by bipolar disorder in both cases. gion, is the causal gene for hereditary neuropathy with The probands and the parents were genotyped with an in- liability to pressure palsies when deleted25 and Charcot- dependent platform both to validate the CNVs and to ex- Marie-Tooth disease type 1A when duplicated.26 In our amine the transmission in the family. We used quad ar- sample, 1 bipolar case had the duplication in this ap- rays (Human 610-quad; Illumina Inc, San Diego, California)

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0.8

0.6

0.4 B Allele Frequency

0.2

0.0 142 895 380 143 539 820 144 184 260 144 828 700 145 473 140 146 117 580 146 762 020 147 406 460 148 050 900

q1 q21.1 q21.2

1 p31.1 q12 q41 q43

eFigure 1. Proband with 1q21.1 duplication. Shown is one of the outputs of the software, which clearly demonstrates the presence of heterozygous single-nucleotide polymorphisms of the type AAB or ABB, ie, 2 alleles from one parent and 1 from the other parent, for the duplicated interval. All findings are supported by normalized intensity values (not shown).

4586766011_R02C02 [8] 1.0

0.8

0.6

0.4 B Allele Frequency

0.2

0.0 142 895 380 143 539 820 144 184 260 144 828 700 145 473 140 146 117 580 146 762 020 147 406 460 148 050 900

q1 q21.1 q21.2

1 p31.1 q12 q41 q43

eFigure 2. Father with identical 1q21.1 duplication. He also had bipolar disorder. following the manufacturer’s protocols (http://www confirmed in the proband but was not found in either .Illumina.com). parent (paternity was confirmed from the SNP data on The 1q21.1 duplication was confirmed in the pro- the array). The father of the proband also had bipolar I band. DNA was available from both parents. The CNV disorder, and both he and his daughter had a similar pre- was also found in the father, who also had bipolar I sentation of illness, with severe manic and depressive epi- disorder. sodes but no psychotic features. One of the probands with the 16p11.2 duplication also The findings from the quad arrays on the 2 families had both parents providing DNA. The duplication was are shown in eFigures 1, 2, 3, 4, 5, and 6.

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0.4 B Allele Frequency

0.2

0.0 142 895 380 143 539 820 144 184 260 144 828 700 145 473 140 146 117 580 146 762 020 147 406 460 148 050 900

q1 q21.1 q21.2

1 p31.1 q12 q41 q43

eFigure 3. Healthy mother of proband with 1q21.1 duplication.

4586766141_R01C01 [13] 1.0

0.8

0.6

0.4 B Allele Frequency

0.2

0.0 28 400 880 28 800 820 29 200 760 29 600 700 30 000 640 30 400 580 30 800 520 31 200 460 31 600 400

1 p13.3 p13.2 p12.3 p12.1 p11 p11.1 q11.2 q12.1 q12.2 q13 q21 q22.1 q22.3 q23.1

eFigure 4. Proband with de novo 16p11.2 duplication.

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0.6

0.4 B Allele Frequency

0.2

0.0 28 400 880 28 800 820 29 200 760 29 600 700 30 000 640 30 400 580 30 800 520 31 200 460 31 600 400

16 p13.3 p13.2 p12.3 p12.1 p11 p11.1 q11.2 q12.1 q12.2 q13 q21 q22.1 q22.3 q23.1

eFigure 5. Father of proband with 16p11.2 duplication (no duplication). He also had bipolar disorder.

4586766017_R02C02 [12] 1.0

0.8

0.6

0.4 B Allele Frequency

0.2

0.0 28 400 880 28 800 820 29 200 760 29 600 700 30 000 640 30 400 580 30 800 520 31 200 460 31 600 400

16 p13.3 p13.2 p12.3 p12.1 p11 p11.1 q11.2 q12.1 q12.2 q13 q21 q22.1 q22.3 q23.1

eFigure 6. Healthy mother of proband with 16p11.2 duplication. Neither parent had the duplication. Single-nucleotide polymorphism analysis showed that the duplication arose from the maternal chromosomes.

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