Influence of Functional Variant of Neuronal Nitric Oxide Synthase on Impulsive Behaviors in Humans

WEB-ONLY CONTENT Influence of Functional Variant of Neuronal Nitric Oxide Synthase on Impulsive Behaviors in Humans

Andreas Reif, MD; Christian P. Jacob, MD; Dan Rujescu, MD; Sabine Herterich, PhD; Sebastian Lang, MD; Lise Gutknecht, PhD; Christina G. Baehne, Dipl-Psych; Alexander Strobel, PhD; Christine M. Freitag, MD; Ina Giegling, MD; Marcel Romanos, MD; Annette Hartmann, MD; Michael Rösler, MD; Tobias J. Renner, MD; Andreas J. Fallgatter, MD; Wolfgang Retz, MD; Ann-Christine Ehlis, PhD; Klaus-Peter Lesch, MD

Arch Gen Psychiatry. 2009;66(1):41-50.

SUPPLEMENTAL METHODS Personality Disorder

A total of 403 inpatients and outpatients of the Department of SAMPLE DESCRIPTIONS Psychiatry and Psychotherapy, University of Würzburg, with personality disorders according to DSM-IV criteria partici- Control A pated in the study (43.2% male; mean [SD] age, 36 [13] years; mean number of Axis II diagnoses, 1.67 [1.15]; Axis I comor- A total of 284 control subjects (150 male and 134 female) con- bidity, 73.2%). Patients were drawn from the general popula- sisting of healthy blood donors originating from Würzburg, Ger- tion of hospital patients; every patient with a clinical diagno- many, were enrolled. The sample was not screened for psychiat- sis of personality disorder treated in the department from 2000 ric disorders; however, all subjects were free of medication, and to 2002 was approached and asked to participate in the study. the study was explained to them, so that the likelihood of severe Inclusion criteria were personality disorder (PD) according to psychiatric disorders in the control sample was low. The mean DSM-IV (antisocial, histrionic, borderline, narcissistic, avoid- (SD) age of controls was 35 (13) years. An additional 356 sub- ant, dependent, and obsessive-compulsive personality disor- jects (174 male and 182 female) were recruited and screened for der) and age between 18 and 60 years. Exclusion criteria were absence of psychiatric disorders by conducting a Structured Clini- medical conditions and lifetime diagnosis of schizophrenia or cal Interview for DSM-IV (SCID-I) interview. These subjects were other psychotic disorders. The sample was divided into sub- recruited by local advertisements, from hospital staff, and from samples of patients with an exclusive diagnosis on either per- medical students. The mean (SD) age of this sample was 30 (9) sonality disorder cluster B or C (ie, cluster B patients with his- years. All participants originated from the catchment area of Lower trionic PD had no cluster C diagnosis). In the cluster B subsample Franconia, a part of Bavaria (southern Germany). (n=322; 43.2% male; mean [SD] age, 35 [13] years), 31.1% had no Axis I diagnosis, whereas F3x (15.2%) and F4x (37.3%) di- Control B agnoses were the 2 most frequent comorbid Axis I disorders. In the cluster C subsample (n=81; 42.0% male; mean [SD] age, Unrelated healthy volunteers of German descent (ie, both of their 38 [13] years; mean [SD] number of Axis II diagnoses, 1.42 parents were German) were randomly selected from the general [0.68]), 12.4% had no Axis I diagnosis, 30.9% had an F3x di- population of Munich, Germany, and contacted by mail. To ex- agnosis, and 42.0% had an F4x diagnosis. Individuals with life- clude subjects with neuropsychiatric disorders or subjects who time diagnosis of schizophrenia or other psychotic disorders had first-degree relatives with neuropsychiatric disorders, we con- were excluded. ducted further screenings before the volunteers were enrolled in The SCID-II was used to diagnose PDs, and PDs were allo- the study. First, subjects who responded were initially screened cated to cluster B and C operationalized as follows: cluster B by telephone. Detailed medical and psychiatric histories were as- (dramatic-emotional) encompasses antisocial, borderline, his- sessed for both themselves and their first-degree relatives by using trionic, and narcissistic PDs. Cluster C (anxious-fearful) in- systematic forms. Second, they were invited to a comprehensive cludes avoidant, dependent, and obsessive-compulsive PDs. The interview including the SCID-I and SCID-II to evaluate their life- assessment of the PDs, including all psychometric testing, was time Axis I and II disorders. Psychiatric diagnoses among their performed by a single experienced psychiatrist (C.P.J.). first-degree relatives were also assessed by means of the Family History Assessment Module.11 Subjects with relevant somatic dis- eases or a lifetime history of any Axis I or II psychiatric disorders Adult Attention-Deficit/Hyperactivity Disorder or suicidal behavior were excluded. Subjects who had first- degree relatives with a lifetime history of a mental disorder or sui- A total of 383 inpatients and outpatients (54.3% male; mean cidal behavior were also excluded. Finally, 1314 healthy sub- [SD] age, 34 [10] years) of the Department of Psychiatry, jects (of whom 601 were male) were included. Their mean (SD) University of Würzburg, referred for diagnostic assessment age was 46 (16) years (range, 19-79 years). and treatment of adult attention-deficit/hyperactivity disor-

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 der (aADHD), were examined with SCID-I. Again, SCID-II cases. The educational and professional status of most of the was used to diagnose PDs, and patients were assessed for subjects was low (special education, school dropout, 13.4%; personality traits by the revised NEO Personality Inventory– junior high school/Յ8 classes, 70.1%; high school/Ͼ8 classes, Revised (NEO-PI-R) Questionnaire. Inclusion criteria were 16.5%; completed professional training, 55.5%; no profes- aADHD according to DSM-IV, onset before the age of 7 sional training, 44.5%), and the rate of unemployment was high years, lifelong persistence, current diagnosis, and age at (employment rate, 39.3%). All subjects underwent a semistruc- recruitment between 18 and 65 years. Exclusion criteria tured psychiatric interview by well-trained psychiatrists (W.R. were a current diagnosis of not withdrawn drug/alcohol and M.R.) and neurologic examination. abuse/dependence, a lifetime diagnosis of bipolar I disorder, schizophrenia, or any other psychotic disorder, and mental Ͻ retardation (IQ 80). Patients were also excluded if the Familial ADHD appearance of symptoms was restricted to the duration of any other Axis I disorder. Details on the sample can be The study included 151 inpatients and outpatients aged 8 to obtained from Jacob et al.12 18 years who had been referred to the Department of Child and Adolescent Psychiatry, University of Würzburg, and Suicide Attempters phenotypically characterized by a team of experienced psychiatrists (C.P.J., M.R., and T.J.R.) in the outpatient unit of Suicide attempters were recruited among patients consecu- the clinic according to DSM-IV criteria. Furthermore, tively referred to general psychiatric wards of the Department included children were characterized by a standardized full of Psychiatry, Ludwig-Maximilians-University, Munich. Only interview (Schedule for Affective Disorders and Schizophre- patients of German descent (ie, both of their parents were Ger- nia for School-Age Children–Present and Lifetime Version man) were enrolled in the study. The sample has been par- [K-SADS-PL]) with a parent. Mothers received (1) the tially published for investigations on other gene variants.13 The unstructured Introductory Interview, (2) the Diagnostic current and lifetime diagnoses of mental disorders were as- Screening Interview, and (3) the Supplement Completion sessed close to discharge by applying the SCID-I interview. Pa- Checklist and, on fulfillment of screening criteria, the appro- tients with mental disorders due to a general medical condi- priate diagnostic supplements. The child was interviewed tion or with dementia were excluded. The study included 189 with the screening interview of the K-SADS and, in cases of suicide attempters (61 of them male) with a clear suicide at- positive screening for mood or anxiety disorders, with the tempt. Their mean (SD) age was 40 (14) years (range, 16-73 respective supplements of the K-SADS-PL. In addition, we years). The DSM-IV lifetime diagnoses of mental disorders among used the Child Behavior Checklist and a German teachers’ the patients were as follows: affective spectrum (n=131), schizo- report on ADHD symptoms according to DSM-IV. phrenia spectrum (n=30), and borderline PD (n=28). The sample included 118 male and 33 female children from 102 families; the mean (SD) age of the affected chil- Forensic dren was 12 (4) years. In all families, both parents were ascertained. The index patient was required to be older than One hundred eighty-two adult male volunteers were enrolled 8 years, and to fulfill DSM-IV criteria for the combined sub- who had been referred for a forensic examination to the Insti- type, other affected siblings in the family had to be older tute of Forensic Psychiatry of the Saarland University, Hom- than 6 years. Exclusion criteria were birth weight less than burg, Germany, by legal authorities for a forensic examination 2000 g, IQ less than 85, neurologic or severe somatic disor- (evaluation of legal responsibility or risk assessment). The mean ders, potentially confounding psychiatric diagnoses such as (SD) age was 34.1 (11.7) years. Criminal offenses of the par- schizophrenia, any pervasive developmental disorder, ticipants of the study comprised homicide, physical injury, and Tourette disorder, primary mood or anxiety disorders, drug robbery (n=75); property offenses and fraud (n=41); sexual of- abuse or autistic disorder, history of any acquired brain fenses (n=33); drug offenses (n=12); and others (n=67). Sub- damage or evidence of the fetal alcohol syndrome, prema- jects with a diagnosis of current substance dependence, acute ture deliveries, and maternal reports of severe prenatal, peri- schizophrenia, major depression/bipolar disorder, or any other natal, or postnatal complications. severe Axis I diagnosis according to DSM-IV as well as men- The forensic sample was ascertained in the southwest tally retarded subjects (IQ, Ͻ70) were not included. Psychiat- German region of Saarland; all other cohorts were recruited ric lifetime diagnoses comprised substance use disorders in Bavaria, a part of southern Germany with a population of (46.2%), psychotic disorders (substance-induced or due to gen- 12.5 million inhabitants. All subjects were white. Only par- eral medical conditions; under stable remission) (8.2%), para- ticipants who gave written informed consent after oral and philias (4.5%), affective disorders (2.2%), and neurotic disor- written explanation of the investigation were enrolled. The ders and disorders of impulse control (3.8%). Cluster B PDs study was approved by the ethics committees of the Univer- were present in 27.2% and PDs of clusters A and C in 7.6% of sity of Würzburg, Homburg, or Munich.

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Gene Symbol Gene Name Fold Change P Value BHLHB2 Basic helix-loop-helix domain containing, class B, 2 1.1348 6.27 ϫ 10−6 GHITM Growth hormone inducible transmembrane protein 1.1308 1.62 ϫ 10−5 HDGFRP3 Hepatoma-derived growth factor, related protein 3 1.1308 5.79 ϫ 10−8 NEFL Neurofilament, light polypeptide 68 kDa 1.1272 1.91 ϫ 10−5 SNCA Synuclein, ␣ (non-A4 component of amyloid precursor) 1.1271 4.83 ϫ 10−6 GRIN1 Glutamate receptor, ionotropic, N-methyl D-aspartate 1 1.1248 3.07 ϫ 10−10 KIAA0182 KIAA0182 protein 1.1226 5.32 ϫ 10−6 WARS Tryptophanyl-tRNA synthetase 1.1216 1.73 ϫ 10−7 GPX3 Glutathione peroxidase 3 (plasma) 1.1214 2.01 ϫ 10−5 TUBB4 Tubulin, ␤,4 1.1197 1.36 ϫ 10−8 SOD2 Superoxide dismutase 2, mitochondrial 1.1148 3.05 ϫ 10−6 RGS4 Regulator of G-protein signaling 4 1.1148 1.06 ϫ 10−5 SNX3 Sorting nexin 3 1.1129 5.29 ϫ 10−7 SRD5A1 Steroid-5-␣-reductase, ␣ polypeptide 1 1.1126 5.17 ϫ 10−6 CACNB2 Calcium channel, voltage-dependent, ␤ 2 subunit 1.1107 5.59 ϫ 10−6 AP1S1 Adaptor-related protein complex 1, ␴ 1 subunit 1.1083 1.42 ϫ 10−5 CLPTM1 Cleft lip and palate associated transmembrane protein 1 1.1082 2.27 ϫ 10−5 OK/SW-cl.56 ␤ 5-Tubulin 1.1072 3.57 ϫ 10−8 DTX3 Deltex 3 homolog (Drosophila) 1.1068 1.67 ϫ 10−7 MEF2C MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C) 1.1066 1.90 ϫ 10−6 TPD52 Tumor protein D52 1.1046 4.60 ϫ 10−6 CHI3L1 Chitinase 3-like 1 (cartilage glycoprotein-39) 1.1016 3.00 ϫ 10−7 MAPK8 Mitogen-activated protein kinase 8 1.1010 2.56 ϫ 10−6 MAP1LC3B Microtubule-associated protein 1 light chain 3 ␤ 1.1008 2.30 ϫ 10−5 YWHAZ Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ␨ polypeptide 1.0985 1.10 ϫ 10−8 APLP2 Amyloid ␤ (A4) precursor-like protein 2 1.0982 1.06 ϫ 10−12 ATP5G3 ATP synthase, Hϩ transporting, mitochondrial F0 complex, subunit c (subunit 9) isoform 3 1.0977 1.15 ϫ 10−5 STAT1 Signal transducer and activator of transcription 1, 91 kDa 1.0973 5.11 ϫ 10−10 PAFAH1B1 Platelet-activating factor acetylhydrolase, isoform Ib, ␣ subunit 45kDa 1.0969 5.46 ϫ 10−6 FEM1B Fem-1 homolog b (Caenorhabditis elegans) 1.0942 1.73 ϫ 10−5 LARP Likely ortholog of mouse la related protein 1.0942 8.92 ϫ 10−7 ATP1B3 ATPase, Naϩ/Kϩ transporting, ␤ 3 polypeptide 1.0938 2.07 ϫ 10−5 NEUROD1 Neurogenic differentiation 1 1.0937 3.35 ϫ 10−6 ID2 Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein 1.0928 3.48 ϫ 10−6 KPNB3 Karyopherin (importin) ␤ 3 1.0928 1.42 ϫ 10−7 HMGN4 High mobility group nucleosomal binding domain 4 1.0918 1.24 ϫ 10−5 DTNA Dystrobrevin, ␣ 1.0908 5.90 ϫ 10−9 ME2 Malic enzyme 2, NADϩ-dependent, mitochondrial 1.0907 3.85 ϫ 10−6 PPP2R5D Protein phosphatase 2, regulatory subunit B (B56), ␦ isoform 1.0903 2.17 ϫ 10−5 ARF3 ADP-ribosylation factor 3 1.0898 3.35 ϫ 10−8 UTY Ubiquitously transcribed tetratricopeptide repeat gene, Y-linked 1.0896 1.89 ϫ 10−5 SFRS2 Splicing factor, arginine/serine-rich 2 1.0895 9.85 ϫ 10−7 FKBP1A FK506 binding protein 1A, 12 kDa 1.0891 1.08 ϫ 10−5 CDC42EP3 CDC42 effector protein (Rho GTPase binding) 3 1.0888 3.22 ϫ 10−7 SNRPB Small nuclear ribonucleoprotein polypeptides B and B1 1.0875 9.44 ϫ 10−6 ACTN2 Actinin, ␣ 2 1.0873 7.91 ϫ 10−6 SRPR Signal recognition particle receptor (“docking protein”) 1.0872 1.20 ϫ 10−5 CUL3 Cullin 3 1.0866 6.31 ϫ 10−8 MAX MAX protein 1.0866 2.14 ϫ 10−7 MCP Membrane cofactor protein (CD46, trophoblast-lymphocyte cross-reactive antigen) 1.0849 5.94 ϫ 10−6 EPB41L3 Erythrocyte membrane protein band 4.1-like 3 1.0849 2.16 ϫ 10−5 HTATIP HIV-1 Tat interactive protein, 60 kDa 1.0842 9.79 ϫ 10−6 SSX2IP Synovial sarcoma, X breakpoint 2 interacting protein 1.0837 1.68 ϫ 10−8 TNFRSF6 Tumor necrosis factor receptor superfamily, member 6 1.0834 5.85 ϫ 10−6 PC4 Activated RNA polymerase II transcription cofactor 4 1.0831 2.40 ϫ 10−5 GFPT1 Glutamine-fructose-6-phosphate transaminase 1 1.0829 1.27 ϫ 10−7 EIF5 Eukaryotic translation initiation factor 5 1.0819 9.22 ϫ 10−11 HLF Hepatic leukemia factor 1.0813 7.69 ϫ 10−6 ING1 Inhibitor of growth family, member 1 1.0812 5.81 ϫ 10−6 ACAT2 Acetyl-coenzyme A acetyltransferase 2 (acetoacetyl Coenzyme A thiolase) 1.0811 1.46 ϫ 10−5 PTPNS1 Protein tyrosine phosphatase, nonreceptor type substrate 1 1.0806 1.69 ϫ 10−5 TDE1 Tumor differentially expressed 1 1.0794 1.18 ϫ 10−9 SRCAP Snf2-related CBP activator protein 1.0783 2.06 ϫ 10−6 GPR51 G protein–coupled receptor 51 1.0779 8.85 ϫ 10−8 SLIT3 Slit homolog 3 (Drosophila) 1.0776 1.26 ϫ 10−5

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 eTable 1. NOS1 Ex1f VNTR Affects the Transcriptome of Human Brodmann Area 46a (continued)

Gene Symbol Gene Name Fold Change P Value LPHN1 Latrophilin 1 1.0770 4.80 ϫ 10−6 ARHGDIA Rho GDP dissociation inhibitor (GDI) ␣ 1.0765 2.46 ϫ 10−8 UBE2D2 Ubiquitin-conjugating enzyme E2D 2 (UBC4/5 homolog, yeast) 1.0765 1.50 ϫ 10−5 MGC3067 Hypothetical protein MGC3067 1.0765 1.63 ϫ 10−5 SLC23A2 Solute carrier family 23 (nucleobase transporters), member 2 1.0764 1.35 ϫ 10−5 SLC7A8 Solute carrier family 7 (cationic amino acid transporter, yϩ system), member 8 1.0761 2.27 ϫ 10−5 C6orf56 Chromosome 6 open reading frame 56 1.0761 1.14 ϫ 10−5 PBX1 Pre–B-cell leukemia transcription factor 1 1.0753 2.00 ϫ 10−6 JUN V-jun sarcoma virus 17 oncogene homolog (avian) 1.0752 3.22 ϫ 10−6 ARFGEF2 ADP-ribosylation factor guanine nucleotide-exchange factor 2 (brefeldin A–inhibited) 1.0752 5.53 ϫ 10−6 FLJ90005 Hypothetical protein FLJ90005 1.0744 8.98 ϫ 10−6 GLS Glutaminase 1.0739 9.53 ϫ 10−6 GRPEL1 GrpE-like 1, mitochondrial (Escherichia coli) 1.0734 7.76 ϫ 10−6 VEGF Vascular endothelial growth factor 1.0734 1.04 ϫ 10−5 BIRC4 Baculoviral IAP repeat-containing 4 1.0733 8.35 ϫ 10−6 WIRE WIRE protein 1.0733 6.43 ϫ 10−6 LOC90379 Hypothetical protein BC002926 1.0729 2.37 ϫ 10−6 PIK4CB Phosphatidylinositol 4-kinase, catalytic, ␤ polypeptide 1.0727 1.36 ϫ 10−5 HSU84971 Vasculogenesis gene on 5q 1.0723 1.03 ϫ 10−6 CAMK2G Calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma 1.0719 3.46 ϫ 10−7 PHTF1 Putative homeodomain transcription factor 1 1.0713 9.37 ϫ 10−6 CNOT4 CCR4-NOT transcription complex, subunit 4 1.0711 2.04 ϫ 10−5 HNRPDL Heterogeneous nuclear ribonucleoprotein D-like 1.0707 1.08 ϫ 10−6 PGK1 Phosphoglycerate kinase 1 1.0705 2.44 ϫ 10−5 FHL1 Four-and-a-half LIM domains 1 1.0695 5.69 ϫ 10−7 NCOA1 Nuclear receptor coactivator 1 1.0695 1.76 ϫ 10−7 PSG6 Pregnancy specific ␤-1-glycoprotein 6 1.0695 4.02 ϫ 10−7 AB026190 Kelch motif containing protein 1.0691 1.41 ϫ 10−6 AGRN Agrin 1.0689 2.39 ϫ 10−5 APC Adenomatosis polyposis coli 1.0687 2.87 ϫ 10−6 HLA-A Major histocompatibility complex, class I, A 1.0683 9.66 ϫ 10−8 CREM cAMP responsive element modulator 1.0680 1.19 ϫ 10−6 PME-1 Protein phosphatase methylesterase 1 1.0674 2.14 ϫ 10−5 SUPT3H Suppressor of Ty 3 homolog (Saccharomyces cerevisiae) 1.0672 7.26 ϫ 10−6 ATF2 Activating transcription factor 2 1.0672 7.85 ϫ 10−6 PSMA1 Proteasome (prosome, macropain) subunit, ␣ type, 1 1.0672 8.32 ϫ 10−7 TNRC9 Trinucleotide repeat containing 9 1.0669 1.88 ϫ 10−5 HSPC056 HSPC056 protein 1.0669 2.47 ϫ 10−5 MTMR1 Myotubularin related protein 1 1.0668 2.44 ϫ 10−5 M11S1 Membrane component, chromosome 11, surface marker 1 1.0667 2.46 ϫ 10−6 CBFA2T2 Core-binding factor, runt domain, ␣ subunit 2; translocated to, 2 1.0658 9.66 ϫ 10−6 PCDH7 BH-protocadherin (brain-heart) 1.0645 4.02 ϫ 10−6 PDE4A Phosphodiesterase 4A, cAMP-specific (phosphodiesterase E2 dunce homolog, Drosophila) 1.0643 1.14 ϫ 10−5 C2orf17 Chromosome 2 open reading frame 17 1.0639 2.87 ϫ 10−6 ABHD6 Abhydrolase domain containing 6 1.0627 8.53 ϫ 10−6 RNF24 Ring finger protein 24 1.0625 1.74 ϫ 10−5 TRIO Triple functional domain (PTPRF interacting) 1.0621 7.63 ϫ 10−6 MALT1 Mucosa associated lymphoid tissue lymphoma translocation gene 1 1.0616 2.36 ϫ 10−5 ATP2C1 ATPase, Caϩϩ transporting, type 2C, member 1 1.0615 1.39 ϫ 10−5 PDE4DIP Phosphodiesterase 4D interacting protein (myomegalin) 1.0613 1.16 ϫ 10−9 ILF3 Interleukin enhancer binding factor 3, 90 kDa 1.0611 7.53 ϫ 10−6 PHLDA1 Pleckstrin homology–like domain, family A, member 1 1.0611 1.71 ϫ 10−9 SNTB2 Syntrophin, ␤ 2 (dystrophin-associated protein A1, 59 kDa, basic component 2) 1.0606 1.22 ϫ 10−5 CYLD Cylindromatosis (turban tumor syndrome) 1.0600 1.49 ϫ 10−6 SSH3BP1 Spectrin SH3 domain binding protein 1 1.0597 7.66 ϫ 10−6 AKAP1 A kinase (PRKA) anchor protein 1 1.0596 2.42 ϫ 10−6 KIAA0368 KIAA0368 1.0596 1.81 ϫ 10−6 FLJ10359 Protein BAP28 1.0594 2.18 ϫ 10−5 MCF2 MCF.2 cell line derived transforming sequence 1.0594 1.39 ϫ 10−6 RPS2 Ribosomal protein S2 1.0589 2.22 ϫ 10−5 PAIP1 Poly(A) binding protein interacting protein 1 1.0584 2.34 ϫ 10−8 PDE10A Phosphodiesterase 10A 1.0578 7.97 ϫ 10−6 RERE Arginine-glutamic acid dipeptide (RE) repeats 1.0576 6.27 ϫ 10−7 ATP5C1 ATP synthase, Hϩ transporting, mitochondrial F1 complex, ␥ polypeptide 1 1.0574 8.95 ϫ 10−6 SNX13 Sorting nexin 13 1.0567 1.77 ϫ 10−5

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Gene Symbol Gene Name Fold Change P Value RNMT RNA (guanine-7-)methyltransferase 1.0567 4.44 ϫ 10−6 KIAA0217 KIAA0217 protein 1.0558 1.69 ϫ 10−5 CAMK2B Calcium/calmodulin-dependent protein kinase (CaM kinase) II ␤ 1.0557 2.75 ϫ 10−6 PPFIA1 Protein tyrosine phosphatase, receptor type, f polypeptide, interacting protein (liprin), ␣ 1 1.0555 1.47 ϫ 10−6 TRRAP Transformation/transcription domain–associated protein 1.0554 1.28 ϫ 10−6 C20orf67 Chromosome 20 open reading frame 67 1.0553 5.33 ϫ 10−6 KIAA0779 KIAA0779 protein 1.0553 1.80 ϫ 10−5 ASAH1 N-acylsphingosine amidohydrolase (acid ceramidase) 1 1.0552 1.19 ϫ 10−5 SNRPA1 Small nuclear ribonucleoprotein polypeptide A’ 1.0552 1.52 ϫ 10−5 GOLGIN-67 Golgin-67 1.0551 2.37 ϫ 10−5 GNAS GNAS complex locus 1.0546 1.58 ϫ 10−5 KPNA1 Karyopherin ␣ 1 (importin ␣ 5) 1.0542 1.80 ϫ 10−6 SET SET translocation (myeloid leukemia-associated) 1.0542 1.75 ϫ 10−5 COPS8 COP9 constitutive photomorphogenic homolog subunit 8 (Arabidopsis) 1.0521 2.32 ϫ 10−5 PSME3 Proteasome (prosome, macropain) activator subunit 3 (PA28 ␥; Ki) 1.0514 3.03 ϫ 10−8 TAF6L TAF6-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 1.0499 1.02 ϫ 10−6 kDa COVA1 Cytosolic ovarian carcinoma antigen 1 1.0485 1.81 ϫ 10−5 WHSC1 Wolf-Hirschhorn syndrome candidate 1 1.0467 6.50 ϫ 10−6 TRO Trophinin 1.0457 1.57 ϫ 10−5 HINT1 Histidine triad nucleotide binding protein 1 1.0455 1.12 ϫ 10−5 DLG3 Disks, large homolog 3 (neuroendocrine-dlg, Drosophila) 1.0454 3.28 ϫ 10−7 GART Phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, 1.0452 1.59 ϫ 10−5 phosphoribosylaminoimidazole synthetase WIZ Widely interspaced zinc finger motifs 1.0448 3.44 ϫ 10−6 PTBP1 Polypyrimidine tract binding protein 1 1.0442 1.09 ϫ 10−5 GAPD Glyceraldehyde-3-phosphate dehydrogenase 1.0442 1.16 ϫ 10−5 SLC35D1 Solute carrier family 35 (UDP-glucuronic acid/UDP-N-acetylgalactosamine dual 1.0440 2.27 ϫ 10−5 transporter), member D1 HGF Hepatocyte growth factor (hepapoietin A; scatter factor) 1.0410 1.40 ϫ 10−5 CFLAR CASP8 and FADD-like apoptosis regulator 1.0407 3.40 ϫ 10−7 RFP Ret finger protein 1.0407 1.76 ϫ 10−5 PMAIP1 Phorbol-12-myristate-13-acetate-induced protein 1 1.0406 8.89 ϫ 10−6 AKAP13 A kinase (PRKA) anchor protein 13 1.0397 2.24 ϫ 10−5 L3MBTL l(3)mbt-like (Drosophila) 1.0393 2.25 ϫ 10−5 NA Similar to homologue of MJD, high homology to a genomic sequence in Xp22 1.0372 2.45 ϫ 10−5 SOS2 Son of sevenless homolog 2 (Drosophila) 1.0359 1.79 ϫ 10−5 EEA1 Early endosome antigen 1, 162 kD 1.0337 7.88 ϫ 10−6 ACTG1 Actin, ␥1 1.0334 4.69 ϫ 10−6 CSHL1 Chorionic somatomammotropin hormone–like 1 1.0171 1.87 ϫ 10−5 LILRB1 Leukocyte immunoglobulin–like receptor, subfamily B (with TM and ITIM domains), −1.0298 2.48 ϫ 10−7 member 1 BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) −1.0538 5.08 ϫ 10−6 SLC6A2 Solute carrier family 6 (neurotransmitter transporter, noradrenalin), member 2 −1.0549 3.91 ϫ 10−6 RPS19 Ribosomal protein S19 −1.0647 2.08 ϫ 10−5 RPL23A Ribosomal protein L23a −1.0758 2.02 ϫ 10−5 GAGE4 G antigen 4 −1.0802 1.62 ϫ 10−5 TXNIP Thioredoxin interacting protein −1.0803 1.60 ϫ 10−6 HBA1 Hemoglobin, ␣ 1 −1.0821 1.17 ϫ 10−6 PBXIP1 Pre–B-cell leukemia transcription factor interacting protein 1 −1.0862 2.80 ϫ 10−6 XIST X (inactive)–specific transcript −1.2185 6.17 ϫ 10−6

Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; cAMP, cyclic adenosine monophosphate; HIV, human immunodeficiency virus; NAD, nicotinamide adenine dinucleotide; NOS1 Ex1f VNTR, variable number tandem repeat polymorphism in the promoter region of exon 1f of nitric oxide synthase 1 (NOS1 ). a Significantly (uncorrected P Ͻ .0000025) dysregulated genes were calculated as a function of NOS1 Ex1f VNTR. The genotypes were analyzed as 0/1/2 values (long-long/short-long/short-short), in which a fold change reported for a gene would be an average fold change for an increase of 1 in the genotype value. Genes are sorted by fold change.

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 eTable 2. Association of History of Violence in 170 Males With NOS1 Ex1f VNTR Genotype and Childhood Adverse Environmental Risk Factors Controlled for Age, History of Substance Use, and History of PDa

Risk Factor Wald ␹2 (df ) P Value Odds Ratio (95% CI) NOS1 genotypes SS vs SL vs LL 6.2 (2) .045 SS vs LL: 3.2 (1.1-9.0) SS vs SL: 1.4 (1.2-7.9) Childhood environmental risk factors 5.1 (1) .02 1.9 (1.1-3.5) No vs history of substance abuse 3.9 (1) .049 0.5 (0.3-1.0) No vs history of personality disorder 0.05 (1) .83 0.9 (0.5-1.9) Age 1.7 (1) .20 0.89 (0.95-1.01) Intercept 0.01 (1) .92

Abbreviations: CI, confidence interval; LL, long-long; NOS1 Ex1f VNTR, variable number tandem repeat polymorphism in the promoter region of exon 1f of nitric oxide synthase 1 (NOS1 ); PD, personality disorder; SL, short-long; SS, short-short. a 2 Test of global null hypothesis: Wald ␹6=15.5, P=.02. Boldface indicates significant results. Associated probability (SD) for violence in NOS1 SS (n=28), 0.57 (0.15); in NOS1 SL (n=96), 0.38 (0.14); and in NOS1 LL (n=46), 0.37 (0.14).

eTable 3. Influence of Sex and NOS1 Ex1f VNTR Genotype on the NEO-PI-R Personality Dimensions in 1099 Healthy Volunteersa

F Scores (Uncorrected P Values) of Analyses of Variance

Effect Neuroticism Extraversion Openness Agreeableness Conscientiousness Sex 4.74 (.03) 7.79 (.005) 1.98 (.16) 5.23 (.02) 8.27 (.004) NOS1 1.11 (.33) 1.63 (.20) 0.37 (.69) 0.19 (.82) 0.67 (.51) NOS1 ϫ sex 0.72 (.49) 0.22 (.80) 0.26 (.77) 0.01 (.99) 6.67 (.001)

Abbreviations: NEO-PI-R, NEO Personality Inventory–Revised; NOS1 Ex1f VNTR, variable number tandem repeat polymorphism in the promoter region of exon 1f of nitric oxide synthase 1 (NOS1 ). a Numerator degrees of freedom are 1 for sex and 2 for all effects involving the NOS1 Ex1f VNTR; effect size: NOS1 ϫ sex, ␩² = 0.01. Boldface indicates PϽ.05.

A Chromosome localization (bp) 116283800 116283900 116284000 116284100 NOS1 UCSC gene predictions based on RefSeq, UniProt, GenBank, and comparative genomics C mRNA Human mRNAs from GenBank Transcripts CpG-rich region CpG islands (islands <300 Bases are light green) Vertebrate multiz alignment and conservation (17 species) Conservation

Mouse

Rat rs3782221 rs9658253 rs11068458 rs1879417 Dog Opossum Chicken Xenopus tropicais Rat (Nov 2004/rn4) chained alignments 13 5 7 Mouse (Feb 2006/mm8) chained alignments Rhesus (Jan 2006/rheMac2) chained alignments Chimp (Mar 2006/panTro2) chained alignments

Repeat Simple tandem repeats by TRF 79

eFigure. Organization and evolutionary conservation of the variable number tandem repeat polymorphism in the promoter region of exon 1f of nitric oxide synthase 1 (NOS1)(NOS1 Ex1f VNTR) region. A, Conservation and genomic structure around NOS1 Ex1f VNTR (region subcloned into reporter gene construct; chromosome 12: 116,283,731 to 116,284,166). B, Black indicates rhesus genome; red, chimp genome; blue, human genome; bold, single-nucleotid polymorphisms; single underlining, species differences; double underlining, repeat region unique to humans (shortened, minimum of 180 repeats in the human genome); dotted underlining, transcribed region (exon 1f ); and arrow, beginning of a CpG dinucleotide island (CpG). C, LD structure around the region (116,280,000 to 116,288,000), as derived from HapMap. bp indicates base pair; mRNA, messenger RNA; TRF, tandem repeat finder; and USCS, University of California, Santa Cruz genome browser.

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