Recurrent Rearrangements in Synaptic and Neurodevelopmental Genes and Shared Biologic Pathways in Schizophrenia, Autism, and Mental Retardation

WEB-ONLY CONTENT Recurrent Rearrangements in Synaptic and Neurodevelopmental Genes and Shared Biologic Pathways in Schizophrenia, Autism, and Mental Retardation

Audrey Guilmatre, PhD; Christèle Dubourg, PhD; Anne-Laure Mosca, MD; Solenn Legallic, BSc; Alice Goldenberg, MD; Vale´rie Drouin-Garraud, MD; Vale´rie Layet, MD; Antoine Rosier, MD; Sylvain Briault, MD; Fre´de´rique Bonnet-Brilhault, MD, PhD; Fre´de´ric Laumonnier, PhD; Sylvie Odent, MD, PhD; Gael Le Vacon, MD; Ge´raldine Joly-Helas, MD; Ve´ronique David, MD; Claude Bendavid, MD; Jean-Michel Pinoit, MD; Ce´line Henry, MD; Caterina Impallomeni, MD; Eva Germano, MD; Gaetano Tortorella, MD; Gabriella Di Rosa, MD; Catherine Barthelemy, MD; Christian Andres, MD; Laurence Faivre, MD, PhD; Thierry Fre´bourg, MD, PhD; Pascale Saugier Veber, PhD; Dominique Campion, MD, PhD

Arch Gen Psychiatry. 2009;66(9):947-956

eTable 1. Polymerase Chain Reaction Primers and Amplification Conditionsa

Gene Forward Primer Reverse Primer Amplicon Size, bp NRXN1␣ exon 1 CGTTAGATAGTCGGGACCCTTATTTCTTCG GATAGGGTTA GCAGCAGGCGTTCCACTT 258 NRXN1␣ exon 5 CGTTAGATAGTAGTGGAGCCTGTGAATGGAAAG GATAGGGTTAGAGGAGCCCTGTATCATGTTGTT 192 NRXN1␤ exon 4 CGTTAGATAGGCCTGGCCCTGCTTTGG GATAGGGTTAGGAAATGGTGGATGTGGTGC 119 DPP10 CGTTAGATAGCAGATAAGTTCCATATTGACTGGG GATAGGGTTAGCTGTTATTTGGTCCTTTACTTCT 180 ERBB4 CGTTAGATAGCCGAGGATGAGTATGTGAATGA GATAGGGTTAGAAGGGTGCTCCGAGGTG 188 CNTN4 CGTTAGATAGGATCTTCAATGAATACCCTTCC GATAGGGTTACCCAGGACCTTGTGGTTTGT 170 GRM7 CGTTAGATAGTGTGAGCCTTGCGATGGTTA GATAGGGTTAGGGCGTGTCATTGTAGCG 245 NLGN1 CGTTAGATAGGATAAGAGGGATTAAGAAGGAACTCA GATAGGGTTAAGGCAGTCTTCGCTCTGGTCT 299 GABRG1 CGTTAGATAGATGACTCCTGGTCTCCATCCT GATAGGGTTAAAGATAGCCAACCCAATAAACCA 278 SLC1A3 CGTTAGATAGGCCAACGAAACGCTTGTG GATAGGGTTAGGAGAAGACAACTAGACCCAGG 158 ST7 CGTTAGATAGCATGTTTTATGGGCATGTGTAC GATAGGGTTAGGAATCTGAGTCCCCGTTAC 236 CNTNAP2 CGTTAGATAGATTTGGGCAATGTGGAGATT GATAGGGTTATTTGGCTCATCTTGGTCTGTG 101 NRG1 CGTTAGATAGTGCGGGAGGCTCAAGGA GATAGGGTTAGGGAAAGAGGCTCGGAA 118 DLGAP2 CGTTAGATAGCAAAAGCAACGCCAACGG GATAGGGTTATGGAGGTCTTGAGGGACAGG 301 PTK2 CGTTAGATAGCCTGGGTACTGGTATGGAACG GATAGGGTTAGTGGTTGGCTCACTATTGCTTT 110 DLG2 CGTTAGATAGAGTAAAGCGGTGGAAGCCCT GATAGGGTTAGCCTTTGAACAGTTTGATTTC 128 GRIP1 CGTTAGATAGACCTTGTGCAGCTCCACAGGA GATAGGGTTATAAAAATTGTCAATGCCAC 277 GABRA5 CGTTAGATAGCAGACCTACCTTCCCTGCATAA GATAGGGTTAAGAGGCAGCACTAGAAGTTTCG 214 APBA2 CGTTAGATAGGAAGCACAAGGGCGAGATC GATAGGGTTAGTGCCTACCTTGATGATGCC 230 CHRNA7 CGTTAGATAG GGACAAGGAGCTGGTCAAGAACT GATAGGGTTA CTGGGGCCAACTAGAGTGC 226 DOC2A CGTTAGATAGCCCTGCAGCCCTCCTTG GATAGGGTTAACTCACTGGCATCATCCGA 100 MAPT CGTTAGATAGAGCCTGGGCGATAGAGCAA GATAGGGTTACTGAGATGCCGTGGAGACG 281 PRODH CGTTAGATAGGGCTTCCCTTTGCCTTTCAG GATAGGGTTAGCCTTACTCCATCTCCTTGTGCT 256 SHANK3 CGTTAGATAGCTCCACCGCAGCCATCTTC GATAGGGTTATGCTCAGACGCACCCAGTT 183 NLGN4 CGTTAGATAGAAGAAGCCCGTCATGGTCTATAT GATAGGGTTATTCCTCTTGCTCAGTCATTCACA 192 TSPAN7 CGTTAGATAGGGATGCTGAAACTGGTGAGTATG GATAGGGTTATTGTTTTTACCTCTAGGGGATTAA 198 CASK CGTTAGATAGGGGTCCCTTCAGTGTTGTACGA GATAGGGTTAATGGAATAAAGCCAGACA 182 IL1RAPL1 CGTTAGATAGAGCCGATGGATGCACTGACT GATAGGGTTACACTTTGGGCAAGGGAGTAAT 148 GRIA3 CGTTAGATAGGCAGTTATACAACACCAACCAGAA GATAGGGTTACTACTTACAAGCATTTGTCACGGA 132 MECP2 CGTTAGATAGTCAAGGACAAACCCCTCAAGT GATAGGGTTAGCCTGACCCTTCTGATGTCTC 166

a Quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) included the following: QMPSF 1 (23 cycles at 94°C for 5 minutes, 94°C for 30 seconds, 57°C for 1 minute, 72°C for 30 seconds, and 72°C for 5 minutes), QMPSF 2 (23 cycles at 94°C for 5 minutes, 94°C for 30 seconds, 56°C for 1 minute, 72°C for 30 seconds, and 72°C for 5 minutes), and QMPSF 3 (24 cycles at 94°C for 5 minutes, 94°C for 30 seconds, 58°C for 1 minute, 72°C for 30 seconds, and 72°C for 5 minutes).

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Chromosomal Case Diagnosis Location Genes Involved Type Confirmation 1003 Control 22q11 PRODH, DGCR6 Dup QMPSF 1060 Control 22q11 PRODH, DGCR6 Dup QMPSF 1069 Control 22q11 PRODH, DGCR6 Dup QMPSF 1136 Control 22q11 PRODH, DGCR6 Dup QMPSF 1182 Control 22q11 PRODH, DGCR6 Dup QMPSF 292/002 Control 22q11 PRODH, DGCR6 Dup QMPSF 1 SZ AFF 22q11 PRODH, DGCR6 Dup QMPSF 99 SZ 22q11 PRODH, DGCR6 Dup QMPSF 136 SZ 22q11 PRODH, DGCR6 Dup QMPSF 189 SZ 22q11 PRODH, DGCR6 Dup QMPSF 236 SZ 22q11 PRODH, DGCR6 Dup QMPSF 244 SZ 22q11 PRODH, DGCR6 Dup QMPSF 280 SZ AFF 22q11 PRODH, DGCR6 Dup QMPSF 44 544 ASDs 22q11 PRODH, DGCR6 Dup QMPSF 44 743 ASDs 22q11 PRODH, DGCR6 Dup QMPSF 44 822 ASDs 22q11 PRODH, DGCR6 Dup QMPSF 46 658 ASDs 22q11 PRODH, DGCR6 Dup QMPSF 11 198 MR 22q11 PRODH, DGCR6 Dup QMPSF 11 984 MR 22q11 PRODH, DGCR6 Dup QMPSF 14 150 MR 22q11 PRODH, DGCR6 Dup QMPSF 14 260 MR 22q11 PRODH, DGCR6 Dup QMPSF 15 051 MR 22q11 PRODH, DGCR6 Dup QMPSF 6355.002 MR 22q11 PRODH, DGCR6 Dup QMPSF 8635 MR 22q11 PRODH, DGCR6 Dup QMPSF 9930 MR 22q11 PRODH, DGCR6 Dup QMPSF 10 403 MR 22q11 PRODH, DGCR6 Dup QMPSF 1050 Control 15q13 CHRNA7 Dup QMPSF, array-CGH 1067 Control 15q13 CHRNA7 Dup QMPSF SDI013 SZ 15q13 CHRNA7 Dup QMPSF 16 144 ASDs 15q13 CHRNA7 Dup QMPSF 14 522 MR 15q13 CHRNA7 Dup QMPSF

Abbreviations: array-CGH, microarray-based technologies for comparative genomic hybridization; ASDs, autism spectrum disorders; Dup, duplication; MR, mental retardation; QMPSF, quantitative multiplex polymerase chain reaction of short fluorescent fragments; SZ, schizophrenia; SZ AFF, schizoaffective disorder.

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Margin, bp

Case CNV Last Outer First Inner Last Inner First Outer Confirmation 113 Dup (Xp11.4) 38 326 267 38 376 483 38 432 836 38 549 143 QMPSF, array-CGH 313 Dup (8p23) 1 436 299 1 481 035 1 590 831 1 642 837 QMPSF, array-CGH 185 Dup (15q13) 26 999 744 27 000 694 27 994 706 28 109 371 QMPSF, array-CCH 144.1 Del (22q11) ...... QMPSF 223 Del (2p16.3) 50 704 195 51 006 556 51 114 057 51 433 167 QMPSF, array-CGH 220 Del (2p16.3) 50 172 024 50 420 164 50 420 164 50 704 195 QMPSF, array-CGH 33 Dup (16p11) ...... QMPSF 136 Dup (16p11) ...... QMPSF 146 Dup (16p11) ...... QMPSF 151 Dup (17q21) 40 869 151 41 073 486 41 700 762 42 143 048 QMPSF, array-CGH T35 Del (2p16.3) 51 006 556 51 114 057 51 114 057 51 433 167 QMPSF, array-CGH, FISH 45 431 Del (2p16.3) 50 704 195 51 006 556 51 114 057 51 433 167 QMPSF, array-CGH 47 604 Del (22q13) 47 124 905 47 265 476 49 525 071 . . . QMPSF, array-CGH Si22 Del (22q13) ...... QMPSF, MLPA 60 478 Dup (15q11-q13) ...... QMPSF, FISH T34 Dup (Xq25) 120 669 107 120 746 477 122 166 142 122 316 448 QMPSF, array-CGH, FISH 12 746 Del (22q11) ...... QMPSF 12 452 Del (22q11) ...... QMPSF 13 899 Del (22q11) ...... QMPSF 44 737 Del (22q11) ...... QMPSF 45 435 Del (22q11) ...... QMPSF 45 856 Del (22q11) ...... QMPSF 46 261 Del (22q11) ...... QMPSF 47 766 Del (22q11) ...... QMPSF Si30 Del (22q11) ...... QMPSF 44 813 Dup (15q13) 26 198 996 26 999 744 30 796 716 30 812 300 QMPSF, array-CGH 12 363 Del (22q11) ...... QMPSF 11 780 Del (22q11) ...... QMPSF 9680 Del (22q11) ...... QMPSF 14 684 Del (22q11) ...... QMPSF 11 695 Del (2p16.3) 51 006 556 51 114 057 51 114 057 51 433 167 QMPSF, array-CGH 14 921 Del (16p11) ...... QMPSF, FISH 10 417 Del (16p11) ...... QMPSF, FISH 13 165 Dup (16p11) ...... QMPSF, FISH 14 390 Del (16p11) ...... QMPSF, FISH 13 907 Del (15q13) 28 109 371 28 725 507 30 298 096 30 701 373 QMPSF, array-CGH 11 919 Del (15q13) 28 109 371 28 725 507 30 298 096 30 701 373 QMPSF, array-CGH 9930 Del (15q13) 28 109 371 28 725 507 30 298 096 30 701 373 QMPSF, array-CGH 12 988 Del (8p23) ...... QMPSF, HRK 011/003 Del (22q11) ...... QMPSF

Abbreviations: array-CGH, microarray-based technologies for comparative genomic hybridization; bp, base pair; Del, deletion; Dup, duplication; ellipsis, not applicable; FISH, fluorescence in situ hybridization; HRK, high-resolution karyotype; MLPA, multiplex ligation-dependent probe amplification; QMPSF, quantitative multiplex polymerase chain reaction of short fluorescent fragments.

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MAPK3 2700

2400 DOC2A

2100

NLGN4 1800 QPRT 1500 PBGD 1200

900

600

300

B 150 180 210 240 270 300

2700

2400 ARVCF USP18 PRODH SNAP29 2100 DGCR6 UFD1L DGCR2 1800

1500 DGSA

1200 MSH2

900

600

300

eFigure 1. A, Detection by a dedicated quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) of a 16p11 duplication. The electropherogram of subject 136 with schizophrenia (red) was superimposed on that of a healthy control subject (blue) by adjusting to the same level the peaks obtained from the amplicon PBGD located on chromosome 11. The vertical axis shows fluorescence in arbitrary units and the horizontal axis indicates the size of the amplicon in base pairs. Horizontal bars indicate heterozygous duplications of the amplicons detected by a 1.5-fold increase of the corresponding peaks. This QMPSF covers 5 genes located at 16p11.2 (DOC2A, QPRT, and MAPK3), Xp22.3 (NLGN4), and 11q23.3 (PBGD). NLGN4 located on the X chromosome was used as a control and shows a 2-fold heightening in the female patient compared with the male control. B, Detection by a dedicated QMPSF of a 22q11 deletion. The electropherogram of subject 9680 with mental retardation (MR) (red) was superimposed on that of a healthy control subject (blue) by adjusting to the same level the peaks obtained from the amplicon MSH2 located on chromosome 2. The vertical axis shows fluorescence in arbitrary units and the horizontal axis indicates the size of the amplicon in base pairs. Horizontal bars indicate heterozygous deletions of the amplicons detected by a 0.5-fold decrease of the corresponding peaks. This QMPSF covers 9 genes located at 22q11 (USP18, PRODH, UFD1L, locus DGSA, ARVCF, DGCR6, SNAP29, and DGCR2) and 2p22 (MSH2). Only PRODH, locus DGSA, and DGCR6 are deleted.

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Schizophrenia

NA Wt NA del 2p16 Wt Wt Mental retardation

1 2 1 2 1 2 Learning disability

del 22q11 del 22q11 del 2p16 del 2p16 del 16p11 Wt Schizoaffective disorder

Family 33 Female

Male NA del 16p11

Deceased

1 2 3 4 5 6 7 8 Sex not indicated

del 16p11 Wt Dup 16p11 Na dup 16p11 NA NA NA

eFigure2. Pedigrees of analyzed multiplex families with copy number variation–bearing probands (arrows). NA indicates not assessed; Wt, wild type; del, deletion; dup, duplication.

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