Association study between gilles de la tourette syndrome and two genes in the robo-slit pathway located in the chromosome 11q24 linkedassociated region.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 147B:68 – 72 (2008) Association Study Between Gilles de la Tourette Syndrome and Two Genes in the Robo-Slit Pathway Located in the Chromosome 11q24 Linked/Associated Region D.M. Miranda,1,2 K. Wigg,2 Y. Feng,2 P. Sandor,2 and C.L. Barr2,3* 1 Department of Pharmacology of Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada 3 Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Ontario, Canada 2 Gilles de la Tourette Syndrome (GTS) is an inherited neuropsychiatric disorder characterized by the presence of motor and phonic tics. Previous genetic studies have identified linkage and association between GTS and the 11q24 chromosomal region. We selected for study, within this region, two possible susceptibility genes for GTS, the ROBO3 and ROBO4 genes. These two genes were selected because of the recent identification of SLITRK1 as a potential susceptibility gene for GTS based on a translocation breakpoint and the further finding of two mutations in the SLITRK1 gene in three patients with GTS. While thus far, the SLITRK1 gene appears to account for only a few cases of GTS, these findings, if confirmed, point to other genes in these pathways that may contribute to GTS. Based on this, we examined two genes in the Slit-Robo pathway involved in cell migration, axonal pathfinding, and/or neuronal differentiation because of their location in 11q24, a region previously identified as linked and associated with GTS. We selected six haplotype tagging single nucleotide polymorphisms (SNPs) for ROBO3 and four for ROBO4 and genotyped them in our sample of trios and sibpair families diagnosed with GTS. Based on 155 nuclear families with 255 affected children, we did not find evidence for association between GTS and either the ROBO3 or ROBO4 genes. Thus, these two genes are unlikely to be the susceptibility genes contributing to GTS on 11q24. ß 2007 Wiley-Liss, Inc. KEY WORDS: Gilles de la Tourette Syndrome; genes; Slit-Robo; axonal pathfinding; association; ROBO3; ROBO4 D.M. Miranda and K. Wigg contributed equally to this work. Grant sponsor: The Tourette Syndrome Association of America; Grant sponsor: NIH; Grant number: MS40024-01; Grant sponsor: Ontario Mental Health Foundation; Grant sponsor: The Tourette Syndrome Foundation of Canada; Grant sponsor: CFRB Radio Station (Toronto). *Correspondence to: C.L. Barr, The Toronto Western Hospital, 399 Bathurst Street, MP14-302, Toronto, Ontario M5T 2S8, Canada. E-mail: CBarr@uhnres.utoronto.ca Received 13 November 2006; Accepted 5 June 2007 DOI 10.1002/ajmg.b.30580 ß 2007 Wiley-Liss, Inc. Please cite this article as follows: Miranda DM, Wigg K, Feng Y, Sandor P, Barr CL. 2008. Association Study Between Gilles de la Tourette Syndrome and Two Genes in the Robo-Slit Pathway Located in the Chromosome 11q24 Linked/Associated Region. Am J Med Genet Part B 147B:68–72. INTRODUCTION Gilles de la Tourette Syndrome (GTS) is a neuropsychiatric disorder characterized by the presence of both motor and phonic tics that begin in childhood and typically wax and wane over periods of weeks or months [Robertson, 2000; Olson, 2004]. Tourette Syndrome usually begins with motor tics at the age of 5–6 years and in most individuals improves in late adolescence and early adulthood [Leckman et al., 1998]. Phonic tics generally begin 1–2 years after the onset of motor tics [Robertson, 2000]. In children, prevalence rates of GTS were previously estimated at 5 per 10,000 with more recent studies providing much higher estimates of 1–3.8% [Robertson, 2003]. Other disorders are commonly observed in affected individuals and in family members of GTS probands [Robertson, 2000]. Obsessive-compulsive symptoms usually co-occur in 20–60% of individuals, but in some studies have been reported as high as 80% [Robertson, 2000]. Symptoms of attentiondeficit hyperactivity disorder (ADHD) are common [Robertson, 2000]. Moreover Tourette’s patients also have elevated rates of depression, personality disorders, anxiety disorders, and social and emotional difficulties [Robertson, 2000; Singer, 2005]. GTS is an inherited disorder, with a high percentage of GTS patients having an affected first-degree relative [Kidd et al., 1980; Hebebrand et al., 1997; Pauls, 2003]. Strong support for the genetic component is provided by evidence from a limited number of twin studies. In a study of 43-twin pairs 53% concordance for Tourette Syndrome diagnosis was observed among monozygotic twins (n ¼ 30 pairs) compared with only 8% among dizygotic twins (n ¼ 13 pairs) [Price et al., 1985]. When criteria were broadened to include any tics, the concordance rates rose to 77% for monozygotic twins and 23% for dizygotic twins [Price et al., 1985]. In a small study of 16 monozygotic twins with one of the pair with GTS, concordance for GTS and tic disorders was 56% and 94%, respectively, with the twin with the lower birth weight having the higher tic score in 12 of the pairs [Hyde et al., 1992]. A number of genome scan and linkage studies have been performed to identify genetic susceptibility loci and several chromosomal regions have been identified with some evidence for linkage [Barr, 2005]. However the LOD scores for these regions are low and not all studies have shown a reproducible linkage region. The difficulty in identifying susceptibility genes, despite evidence for a strong genetic contribution to ROBO3/ROBO4 and Tourette Syndrome GTS, has been attributed to uncertainties about the mode of inheritance, difficulty defining the exact phenotype for genetic studies and locus heterogeneity [Barr and Sandor, 1998; Barr et al., 1999; Barr, 2005]. One locus that has shown some reproducibility is the 11q24 region. A case-control study by Simonic et al. , using 100 cases and 96 controls from the Afrikaner population, found association between GTS and the 11q region as well as three additional chromosomal regions. The 11q region was originally identified as 11q23 however results from the human genome project indicate that the location of the most significant markers were in 11q24 (http://genome.ucsc.edu/cgi-bin/hgGateway?org¼human). In a subsequent study, this group confirmed the finding of association with the 11q24 chromosomal region, particularly with the marker D11S1377 in an independent sample of 91 nuclear families with one or more affected children using family based association controls (haplotype relative risk and the transmission disequilibrium test) [Simonic et al., 2001]. In the study of Merette et al. , evidence for linkage was tested in a large French Canadian family (127 members) with members diagnosed with GTS or isolated tics. Significant evidence for linkage was found for the markers D11S1377 and D11S933. However, an association study of the same region of chromosome 11q24 with 6 markers in 199 French–Canadian nuclear families, 175 trios plus 25 incomplete trios, failed to find association with the markers D11S1377 or D11S933 [Diaz-Anzaldua et al., 2005]. While both the Simovic and Diaz-Anzaldua studies used association analyses that rely on linkage disequilibrium, the Afrikaner population has been shown to have longer regions of linkage disequilibrium; on average 6 cM compared to 3 cM in other founder and outbred populations [Hall et al., 2002]. Thus, the region of linkage disequilibrium across this region is likely to be stronger in the Afrikaner study allowing for the identification of a susceptibility gene at twice the distance from the markers compared to other populations. The amount of linkage disequilibrium between the markers in the Quebec study was very low, thus the marker density across the region may have been insufficient to detect association. Based on the position in the linked/associated region of 11q24, we targeted two genes as promising candidates— roundabout3 (ROBO3) and roundabout4 (ROBO4). These two genes are attractive candidates because of their involvement in the Slit-Robo pathway, a system involved in axonal pathfinding, neuronal migration, and dendritic branching [Zhu et al., 1999; Bagri et al., 2002; Mambetisaeva et al., 2005]. Changes in these processes as a mechanism contributing to GTS is supported by the recent finding of Abelson et al. . That study found one child with GTS and a de novo inversion on chromosome 13, with a breakpoint close to the gene encoding Slit and Trk-like 1 (SLITRK1). This gene is a member of a gene family implicated in neurite outgrowth [Aruga and Mikoshiba, 2003]. Based on the translocation finding, this gene was screened in 174 unrelated subjects with GTS and two mutations in this gene were identified in three unrelated individuals: (1) a frame shift mutation that results in the substitution of 27 amino acids and a premature termination and (2) a mutation in the 30 untranslated region within a predicted binding site for a microRNA (var321). These mutations were not found in 3,600 control chromosomes. Both mutations are predicted to result in haploinsufficiency. Primary neuronal cultures taken from embryos transfected with wild type SLITRK1 were shown to have enhanced dendritic growth, but neurons from embryos transfected with the SLITRK1 frameshift mutant did not [Abelson et al., 2005]. SLITRK1 is expressed widely in developing and postnatal brain and the implication of a protein regulating neuronal development open a new line of inquiry into the genetic susceptibility to GTS. 69 The Robo family of proteins are transmembrane receptors for the Slit proteins [Brose et al., 1999]. The function of the Robo proteins was first elucidated in Drosophila where it was noted that mutations created increased axonal crossing and recrossing of the ventral midline [Seeger et al., 1993; Kidd et al., 1999]. Continued studies of these proteins in Drosophila and vertebrates indicate that it is the precise temporal and spatial expression, as well as the relative ratios of the Robo and Slit proteins (as well as other proteins), that create chemorepulsive and chemoattaction properties. These combined effects result in the correct alignment of axons at the midline, neuronal differentiation, tangential neuronal migration in the neocortex, and the correct development of the cortifugal, callosal, and thalmocorticol tracts [Zhu et al., 1999; Bagri et al., 2002; Connor and Key, 2002; Whitford et al., 2002; Mambetisaeva et al., 2005]. Although the Slit-Robo proteins are best known for their role in axonal pathfinding and neuronal migration, a more general role in cell migration has become apparent [Wong et al., 2002; Kaur et al., 2006]. Of interest is evidence for a role in lymphocyte chemotaxis [Wu et al., 2001]. Given some evidence, although still controversial, for an autoimmune mechanism as a contributing factor in GTS [Kiessling et al., 1993; Singer et al., 1998; Hallett et al., 2000; Taylor et al., 2002; Leckman et al., 2005], susceptibility genes that regulate immune function as well as neuronal development are attractive candidates. The Robo gene family is composed of 4 genes, ROBO1, ROBO2, ROBO3, and the recently identified ROBO4. These genes are located in the chromosomal regions 3p12.3 for ROBO1 and ROBO2, and 11q24.2 for ROBO3 and ROBO4. Interestingly, ROBO1 has been reported as a gene contributing to developmental dyslexia (specific reading disabilities) [Hannula-Jouppi et al., 2005]. The evidence in support of this is deduced from a chromosomal translocation identified in the first intron of ROBO1 in a single individual however two other family members with dyslexia do not carry the translocation [Hannula-Jouppi et al., 2005]. Evidence was also derived from a single large pedigree linked to this region of 3p where a haplotype of the gene segregates with low expression of the ROBO1 gene as measured in Epstein–Barr transformed lymphocytes [Hannula-Jouppi et al., 2005]. While the indication of a gene regulating axonal pathfinding and neuronal differentiation as a contributor to a specific learning disability is intriguing, at the current time the evidence supporting ROBO1 as a contributor to dyslexia is equivocal and requires replication. The creation of mice deficient in Robo3 (Rig-1 in mice) indicates that Robo3 functions to repress responsiveness to Slit in the growth cone until after midline crossing [Sabatier et al., 2004]. Several possible mechanisms for this have been proposed including a role for Robo3 in masking the repellant properties of Slit, possibly by preventing Robo1 from reaching the membrane or by Robo3 acting as a sink for Slit binding [Guthrie, 2004]. ROBO3 mutations have been identified in patients with the syndrome of horizontal gaze palsy (HGPPS; Online Mendelian Inheritance in Man, OMIM 607313), a rare human disorder characterized by the absence of conjugate horizontal eye movement and progressive scoliosis. Using high-resolution magnetic resonance imaging, Jen et al.  found an error in midline crossing of the ascending sensory and descending motor pathways in the medulla. All 10 individuals with HGPPS tested in that study were from consanguineous families and were homozygous for mutations in ROBO3. Nine of the 10 had mutations in the extracellular domain and the 10th had an insertion nucleotide that predicts a premature termination of the protein. This finding indicates that Robo3 is required for hindbrain axon midline crossing in humans as has been demonstrated in mice [Sabatier et al., 2004; Mambetisaeva et al., 2005]. 70 Miranda et al. ROBO4 or magic roundabout was identified in a search for endothelial specific transcripts using bioinformatics techniques and highest expression was found in placenta, umbilical cord, and some neoplastic tumors [Huminiecki et al., 2002]. In that study using RNAase protection assays and immunohistochemistry, expression of ROBO4 was detected in endothelial cell lines, but not a neuroblastoma cell line [Huminiecki et al., 2002]. Using in situ hybridization, expression was observed in placenta, umbilical cord tissue, vessels in the bladder, and breast carcinoma, however expression was not detected in brain cerebrum. A screen using gene-specific primers showed ROBO4 to be present in libraries from a number of tissues including adult and fetal brain. The authors conclude from the expression pattern that ROBO4 is endothelial specific, however data available from public databases (www.geneatlas.org; www.brain-map.org) indicate that ROBO4 is expressed in brain. Further, expression patterns in developing zebrafish indicate expression in both the neural tube and the central nervous system [Bedell et al., 2005]. ROBO4 is the most recent gene identified in the Slit-Robo pathway and thus less is known of the role of this protein. Robo4 is the predominant Robo protein expressed during the development of the embryonic vasculature in zebrafish and a critical role in vascular guidance has been identified [Bedell et al., 2005]. Current data indicates that the relative levels of Robo1 and Robo4 guide the growing tip of endothelial cells to their target through response to environmental signals much as the Robo proteins do for axons [Bedell et al., 2005]. On the basis of the SLITRK1 findings, we hypothesized that genes of the Slit-Robo pathway may contribute to susceptibility to GTS. Based on linkage and association findings on11q24, we targeted the ROBO3 and ROBO4 genes for study. MATERIALS AND METHODS Subjects Our sample consisted of 155 nuclear families, including both affected sibling pair families and parent/proband trios with a total of 255 affected children recruited from the Tourette Syndrome Clinic of The Toronto Western Hospital. Written informed parental consent and verbal assent for younger children or written patient consent was obtained for all participants and the study was approved by the research ethics committee of the University Health Network. Information about symptoms associated with GTS and obsessive compulsive disorder were collected in a two-stage process, first using selfand family-report based on the tic inventory and ordinal severity scales of the Yale Global Tic Severity Scale [Leckman et al., 1989] and the symptom checklist and ordinal scales of the Yale-Brown Obsessive-compulsive scale [Goodman et al., 1989]. TABLE I. TDT Analysis of the ROBO3 Gene Polymorphism rs11219819 rs4936957 rs3923890 rs11219821 rs4606490 rs3802905 Allele Allele frequency Transmissions Non-transmission w2 P-value C T C T A T C T C T C G 0.757 0.243 0.67 0.326 0.310 0.690 0.545 0.455 0.446 0.554 0.620 0.380 62 52 78 69 76 69 89 84 83 82 81 77 52 62 69 78 69 76 84 89 82 83 77 81 0.877 0.349 0.551 0.457 0.338 0.561 0.145 0.703 0.006 0.938 0.101 0.750 TABLE II. TDT of the ROBO4 Gene Polymorphism rs4078483 rs4635093 rs6590109 rs12823 Allele Allele frequency Transmissions Non-transmission w2 P-value C G G T A G A T 0.306 0.694 0.298 0.702 0.667 0.333 0.350 0.650 44 42 33 30 55 46 37 30 42 44 30 33 46 55 30 37 0.048 0.826 0.143 0.705 0.516 0.472 0.731 0.392 TABLE III. Linkage Disequilibrium Between Markers Used in the Study of ROBO3 rs11219821 rs11219821 rs4936957 rs3923890 rs11219821 rs4606490 rs3802905 0.05 0.07 0.10 0.04 0.16 rs4936957 rs3923890 rs11219821 rs4606490 rs3802905 0.27 0.30 0.92 0.60 0.96 0.96 0.32 0.83 0.97 0.97 0.88 0.87 0.93 0.88 0.91 0.78 0.37 0.43 0.23 0.34 0.53 0.24 2 0.61 0.58 D0 -values are shown in the upper half of the table, r in the lower half of the table. 0.41 ROBO3/ROBO4 and Tourette Syndrome 71 TABLE IV. Linkage Disequilibrium Between Markers Used in the Study of ROBO4 rs4078483 rs4078483 rs4635093 rs6590109 rs12823 0.19 0.84 0.22 rs4635093 rs6590109 rs12823 0.97 0.99 1.0 0.98 0.98 0.98 0.21 0.23 0.26 2 0 D -values are shown in the upper half of the table, r in the lower half of the table. In a second stage, the information was checked by an experienced neuropsychiatrist, who also performed a direct examination of each proband and sibling included in this study. strong linkage disequilibrium across these genes in our sample as observed in the HapMap data set (www.hapmap.org). Genotyping DISCUSSION Blood was collected from the subjects and genomic DNA was extracted from leukocytes using a standard high salt extraction method [Miller et al., 1988]. A total of 10 markers from ROBO3 and ROBO4 genes were genotyped using the TaqMan1 assay with primer and probes available commercially (Applied Biosystems, Inc., ABI, Foster, CA). The PCR primers and allelic discrimination probes were obtained from ABI (Assay-On-Demand). Five microliters of PCR reactions contained 30 ng of genomic DNA, 5 mmol of TaqMan1 Universal PCR Master Mix (Applied Biosystems) and 0.125 ml of allelic discrimination mix, which contained 18 mM of each primer and 4 mM of each probe. The thermal cycling conditions for all these primers were 958C for 10 min, then 40–60 cycles of 928C for 15 sec and 1 min at 598C. Negative controls were included with each 96-well plate. Plates were read on the ABI 7900-HT Sequence Detection System1 (Applied Biosystems) using the allelic discrimination endpoint analysis mode of the software package version 2.0 (Applied Biosystems). For the categorical analysis of GTS the TDT statistic implemented in the ETDT program [Sham and Curtis, 1995] was used to test for the biased transmission of alleles to affected children. The coefficients of linkage disequilibrium between markers alleles, D2 and D0 were calculated using Haploview v2.03 [Barrett et al., 2005]. The transmission of haplotypes was analyzed using the TRANSMIT program using the robust estimator of variance option [Clayton, 1999]. In the past 15 years many strategies have been used to identify susceptibility genes for GTS including genome scans and candidate genes studies. Several genome scans were performed previously and a number of chromosomal regions have been identified as suggestive of linkage, but the LOD scores for these loci were, for the most part, low and these studies have failed to show consistent findings across all studies [Barr, 2005]. A recent genome scan with a large sample of 304 affected sibling pairs provides significant evidence for linkage to chromosome 2p (The Tourette Syndrome Association International Consortium for Genetics, 2007). The finding of two different mutations in the SLITRK1 gene in three individuals with GTS may be the first possible clue to the understanding of the biological basis underlying the genetic susceptibility to GTS. However, at this time mutations in the SLITRK1 gene appear to contribute to only a very small number of cases and this finding has not as yet been replicated in additional studies [Verkerk et al., 2006; Wendland et al., 2006]. Our study is the first study to investigate genes in the SlitRobo pathway in GTS. These two genes were chosen principally based on their location in the chromosomal 11q24 linked/ associated region and are located 2 Mbp from the most significant marker (D11S1377, Mfd316) associated in the Simonic et al.  study and 63 kb away from D11S933, a marker supported in the Simonic case-control Afrikaner study [Simonic et al., 1998]. While we failed to find association between the ROBO3 and ROBO4 genes with GTS in this study, we cannot rule out other genes influencing the Slit-Robo pathway in the genetic susceptibility of GTS. RESULTS ACKNOWLEDGMENTS To investigate the association between GTS and the ROBO3 and ROBO4 genes, we genotyped four markers in the ROBO4 gene and six markers in the ROBO3 gene in a sample of 155 families with 255 affected individuals. These markers were chosen to tag the major haplotypes to ensure adequate coverage of each gene. We used the Tagger Pairwise Method as implemented on the International HapMap Project Browser (www.hapmap.org) to select haplotype tagging single nucleotide polymorphisms (SNPs). The SNPs selected for both ROBO3 and ROBO4 met the parameters of an r2 value greater than 0.7 and a minor allele frequency of 0.20. All markers genotyped for this manuscript were haplotype tagging SNPs. The results of the TDT analysis for the markers used in this study are shown in Table I for ROBO3 and Table II for ROBO4. We found no evidence for biased transmission of any of the alleles for either gene to affected children. Further, we did not observe statistical evidence for biased transmission of haplotypes for any of the markers in our sample of GTS families (not shown). Haplotype analyses were performed using the four markers in ROBO4 and separately for the six markers in ROBO3. The linkage disequilibrium between the markers was estimated using Haploview (Tables III and IV). We observed This work was supported by a Doctoral Fellowship from the CAPES/Brazil to Dr. Miranda. The collection of families for this study was supported by grants from The Tourette Syndrome Association of America, NIH grant MS40024-01, the Ontario Mental Health Foundation, The Tourette Syndrome Foundation of Canada, and CFRB radio station (Toronto). Statistical Analysis REFERENCES Abelson JF, Kwan KY, O’Roak BJ, Baek DY, Stillman AA, Morgan TM, Mathews CA, Pauls DL, Rasin MR, Gunel M, et al. 2005. Sequence variants in SLITRK1 are associated with Tourette’s syndrome. Science 310(5746):317–320. Aruga J, Mikoshiba K. 2003. Identification and characterization of Slitrk, a novel neuronal transmembrane protein family controlling neurite outgrowth. Mol Cell Neurosci 24(1):117–129. Bagri A, Marin O, Plump AS, Mak J, Pleasure SJ, Rubenstein JL, TessierLavigne M. 2002. Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron 33(2):233–248. Barr CL. 2005. Progress in Gene Localization. In: Kurlan R, editor. Handbook of Tourette’s Syndrome and Related Tic and Behavioral Disorders. New York, NY: Marcel Dekker Inc. p 379–398. 72 Miranda et al. Barr CL, Sandor P. 1998. Current status of genetic studies of Gilles de la Tourette syndrome. Can J Psychiatry 43(4):351–357. Barr CL, Wigg KG, Pakstis AJ, Kurlan R, Pauls D, Kidd KK, Tsui LC, Sandor P. 1999. Genome scan for linkage to Gilles de la Tourette syndrome. Am J Med Genet 88(4):437–445. Barrett JC, Fry B, Maller J, Daly MJ. 2005. Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics 21(2):263–265. Bedell VM, Yeo SY, Park KW, Chung J, Seth P, Shivalingappa V, Zhao J, Obara T, Sukhatme VP, Drummond IA, et al. 2005. roundabout4 is essential for angiogenesis in vivo. Proc Natl Acad Sci U S A 102(18): 6373–6378. Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS, TessierLavigne M, Kidd T. 1999. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96(6): 795–806. Leckman JF, Katsovich L, Kawikova I, Lin H, Zhang H, Kronig H, Morshed S, Parveen S, Grantz H, Lombroso PJ, et al. 2005. Increased serum levels of interleukin-12 and tumor necrosis factor-alpha in Tourette’s syndrome. Biol Psychiatry 57(6):667–673. Mambetisaeva ET, Andrews W, Camurri L, Annan A, Sundaresan V. 2005. Robo family of proteins exhibit differential expression in mouse spinal cord and Robo-Slit interaction is required for midline crossing in vertebrate spinal cord. Dev Dyn 233(1):41–51. Merette C, Brassard A, Potvin A, Bouvier H, Rousseau F, Emond C, Bissonnette L, Roy MA, Maziade M, Ott J, et al. 2000. Significant linkage for Tourette syndrome in a large French Canadian family. Am J Hum Genet 67(4):1008–1013. Miller SA, Dykes DD, Polesky HF. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16(3):1215. Clayton D. 1999. A Generalization of the Transmission/Disequilibrium Test for Uncertain-Haplotype Transmission. Am J Hum Genet 65(4):1170–1177. Olson S. 2004. Neurobiology. Making sense of Tourette’s. Science 305(5689): 1390–1392. Connor RM, Key B. 2002. Expression and role of Roundabout-1 in embryonic Xenopus forebrain. Dev Dyn 225(1):22–34. Pauls DL. 2003. An update on the genetics of Gilles de la Tourette syndrome. J Psychosom Res 55(1):7–12. Diaz-Anzaldua A, Riviere JB, Dube MP, Joober R, Saint-Onge J, Dion Y, Lesperance P, Richer F, Chouinard S, Rouleau GA. 2005. Chromosome 11-q24 region in Tourette syndrome: Association and linkage disequilibrium study in the French Canadian population. Am J Med Genet Part A 138:225–228. Price RA, Kidd KK, Cohen DJ, Pauls DL, Leckman JF. 1985. A twin study of Tourette syndrome. Arch Gen Psychiatry 42(8):815–820. Goodman WK, Price LH, Rasmussen SA, Mazure C, Fleischmann RL, Hill CL, Heninger GR, Charney DS. 1989. The Yale-Brown Obsessive Compulsive Scale. I. Development, use, and reliability. Arch Gen Psychiatry 46(11):1006–1011. Guthrie S. 2004. Axon guidance: Mice and men need Rig and Robo. Curr Biol 14(15):R632–R6324. Hall D, Wijsman EM, Roos JL, Gogos JA, Karayiorgou M. 2002. Extended intermarker linkage disequilibrium in the Afrikaners. Genome Res 12(6):956–961. Hallett JJ, Harling-Berg CJ, Knopf PM, Stopa EG, Kiessling LS. 2000. Antistriatal antibodies in Tourette syndrome cause neuronal dysfunction. J Neuroimmunol 111(1–2):195–202. Hannula-Jouppi K, Kaminen-Ahola N, Taipale M, Eklund R, Nopola-Hemmi J, Kaariainen H, Kere J. 2005. The Axon Guidance Receptor Gene ROBO1 Is a Candidate Gene for Developmental Dyslexia. PLoS Genet 1(4):e50. Hebebrand J, Klug B, Fimmers R, Seuchter SA, Wettke-Schafer R, Deget F, Camps A, Lisch S, Hebebrand K, von Gontard A, et al. 1997. Rates for tic disorders and obsessive compulsive symptomatology in families of children and adolescents with Gilles de la Tourette syndrome. J Psychiatr Res 31(5):519–530. Huminiecki L, Gorn M, Suchting S, Poulsom R, Bicknell R. 2002. Magic roundabout is a new member of the roundabout receptor family that is endothelial specific and expressed at sites of active angiogenesis. Genomics 79(4):547–552. Hyde TM, Aaronson BA, Randolph C, Rickler KC, Weinberger DR. 1992. Relationship of birth weight to the phenotypic expression of Gilles de la Tourette’s syndrome in monozygotic twins. Neurology 42(3 Pt 1):652– 658. Jen JC, Chan WM, Bosley TM, Wan J, Carr JR, Rub U, Shattuck D, Salamon G, Kudo LC, Ou J, et al. 2004. Mutations in a human ROBO gene disrupt hindbrain axon pathway crossing and morphogenesis. Science 304(5676):1509–1513. Kaur S, Castellone MD, Bedell VM, Konar M, Gutkind JS, Ramchandran R. 2006. Robo4 signaling in endothelial cells implies attraction guidance mechanisms. J Biol Chem 281(16):11347–11356. Kidd KK, Prusoff BA, Cohen DJ. 1980. Familial pattern of Gilles de la Tourette syndrome. Arch Gen Psychiatry 37(12):1336–1339. Kidd T, Bland KS, Goodman CS. 1999. Slit is the midline repellent for the RoBo receptor in Drosophila. Cell 96(6):785–794. Kiessling LS, Marcotte AC, Culpepper L. 1993. Antineuronal antibodies in movement disorders. Pediatrics 92(1):39–43. Leckman JF, Riddle MA, Hardin MT, Ort SI, Swartz KL, Stevenson J, Cohen DJ. 1989. The Yale Global Tic Severity Scale: Initial testing of a clinicianrated scale of tic severity. J Am Acad Child Adolesc Psychiatry 28(4):566–573. Leckman JF, Zhang H, Vitale A, Lahnin F, Lynch K, Bondi C, Kim YS, Peterson BS. 1998. Course of tic severity in Tourette syndrome: The first two decades. Pediatrics 102(1 Pt 1):14–19. Robertson MM. 2000. Tourette syndrome, associated conditions and the complexities of treatment. Brain 123(Pt 3):425–462. Robertson MM. 2003. Diagnosing Tourette syndrome: Is it a common disorder? J Psychosom Res 55(1):3–6. Sabatier C, Plump AS, Le M, Brose K, Tamada A, Murakami F, Lee EY, Tessier-Lavigne M. 2004. The divergent Robo family protein rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons. Cell 117(2):157–169. Seeger M, Tear G, Ferres-Marco D, Goodman CS. 1993. Mutations affecting growth cone guidance in Drosophila: Genes necessary for guidance toward or away from the midline. Neuron 10(3):409–426. Sham PC, Curtis D. 1995. An extended transmission/disequilibrium test (TDT) for multi-allele marker loci. Ann Hum Genet 59(Pt 3):323–336. Simonic I, Gericke GS, Ott J, Weber JL. 1998. Identification of genetic markers associated with Gilles de la Tourette syndrome in an Afrikaner population. Am J Hum Genet 63(3):839–846. Simonic I, Nyholt DR, Gericke GS, Gordon D, Matsumoto N, Ledbetter DH, Ott J, Weber JL. 2001. Further evidence for linkage of Gilles de la Tourette syndrome (GTS) susceptibility loci on chromosomes 2p11, 8q22 and 11q 23-24in South African Afrikaners. Am J Med Genet 105(2):163– 167. Singer HS. 2005. Tourette’s syndrome: From behaviour to biology. Lancet Neurol 4(3):149–159. Singer HS, Giuliano JD, Hansen BH, Hallett JJ, Laurino JP, Benson M, Kiessling LS. 1998. Antibodies against human putamen in children with Tourette syndrome. Neurology 50(6):1618–1624. Taylor JR, Morshed SA, Parveen S, Mercadante MT, Scahill L, Peterson BS, King RA, Leckman JF, Lombroso PJ. 2002. An animal model of Tourette’s syndrome. Am J Psychiatry 159(4):657–660. The Tourette Syndrome Association International Consortium for Genetics. 2007. Genome scan for tourette disorder in affected-sibling-pair and multigenerational families. Am J Hum Genet 80(2):265–272. Verkerk AJ, Cath DC, van der Linde HC, Both J, Heutink P, Breedveld G, Aulchenko YS, Oostra BA. 2006. Genetic and clinical analysis of a large Dutch Gilles de la Tourette family. Mol Psychiatry 11(10):954– 964. Wendland JR, Kruse MR, Murphy DL. 2006. Functional SLITRK1 var321, varCDfs and SLC6A4 G56A variants and susceptibility to obsessivecompulsive disorder. Mol Psychiatry 11(9):802–804. Whitford KL, Marillat V, Stein E, Goodman CS, Tessier-Lavigne M, Chedotal A, Ghosh A. 2002. Regulation of cortical dendrite development by Slit-Robo interactions. Neuron 33(1):47–61. Wong K, Park HT, Wu JY, Rao Y. 2002. Slit proteins: Molecular guidance cues for cells ranging from neurons to leukocytes. Curr Opin Genet Dev 12(5):583–591. Wu JY, Feng L, Park HT, Havlioglu N, Wen L, Tang H, Bacon KB, Jiang Z, Zhang X, Rao Y. 2001. The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature 410(6831):948–952. Zhu Y, Li H, Zhou L, Wu JY, Rao Y. 1999. Cellular and molecular guidance of GABAergic neuronal migration from an extracortical origin to the neocortex. Neuron 23(3):473–485.