Psychiatry Research 165 (2009) 1–9 www.elsevier.com/locate/psychres Polymorphisms in genes involved in neurodevelopment may be associated with altered brain morphology in schizophrenia: Preliminary evidence Sheila P. Gregório a,b, Paulo C. Sallet a, Kim-Anh Do c, E. Lin c, ⁎ Wagner F. Gattaz a, Emmanuel Dias-Neto a, ,1 a Laboratório de Neurociências (LIM-27), Departmento e Instituto de Psiquiatria, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil b Departmento de Bioquímica, Inst. Química, Universidade de São Paulo, São Paulo, Brazil c Department of Biostatistics, the University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd, 77030, Houston, TX, USA Received 24 June 2006; received in revised form 16 July 2007; accepted 18 August 2007 Abstract An abnormality in neurodevelopment is one of the most robust etiologic hypotheses in schizophrenia (SZ). There is also strong evidence that genetic factors may influence abnormal neurodevelopment in the disease. The present study evaluated in SZ patients, whose brain structural data had been obtained with magnetic resonance imaging (MRI), the possible association between structural brain measures, and 32 DNA polymorphisms, located in 30 genes related to neurogenesis and brain development. DNA was extracted from peripheral blood cells of 25 patients with schizophrenia, genotyping was performed using diverse procedures, and putative associations were evaluated by standard statistical methods (using the software Statistical Package for Social Sciences - SPSS) with a modified Bonferroni adjustment. For reelin (RELN), a protease that guides neurons in the developing brain and underlies neurotransmission and synaptic plasticity in adults, an association was found for a non-synonymous polymorphism (Val997Leu) with left and right ventricular enlargement. A putative association was also found between protocadherin 12 (PCDH12), a cell adhesion molecule involved in axonal guidance and synaptic specificity, and cortical folding (asymmetry coefficient of gyrification index). Although our results are preliminary, due to the small number of individuals analyzed, such an approach could reveal new candidate genes implicated in anomalous neurodevelopment in schizophrenia. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Polymorphisms; Magnetic resonance imaging (MRI); Single nucleotide polymorphism (SNP); Brain morphometry; Reelin; PCDH12 1. Introduction There is consistent evidence that schizophrenia (SZ) ⁎ Corresponding author. Lab. of Neurosciences (LIM-27), Instituto is a neurodevelopmental disorder. A number of twin and de Psiquiatria Faculdade de Medicina, Universidade de São Paulo. R. family studies point to a genetic basis for SZ, involving Dr. Ovidio Pires de Campos, 785-3o andar-Consolação, 05403-010, several genes and in many chromosomal regions (Kohn São Paulo, SP, Brazil. Tel.: +55 11 3069.7267; fax: +55 11 3069.8011. E-mail address: [email protected] (E. Dias-Neto). and Lerer, 2002; McGuffin et al., 2003), in conformity 1 Present address: University of Texas, MD Anderson Cancer with complex-polygenic diseases. It has been suggested Center, 1515 Holcombe Blvd, 77030, Houston, TX, USA. that nearly 30% of the human genes (many of which are 0165-1781/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.psychres.2007.08.011 2 S.P. Gregório et al. / Psychiatry Research 165 (2009) 1–9 developmentally regulated) are expressed in the brain Table 1 (Kozlovsky et al., 2002). Many of these genes are Demographic and clinical characteristics of SZ patients evaluated by MRI scans and genotyping located in chromosomal loci associated with SZ and are potential candidate genes due to their polymorphic sta- Characteristic Schizophrenic tus in the population and/or to events that alter their patients (N=25) expression during embryonic stages, which might result N % in the putative neurodevelopmental abnormalities seen Gender Male 15 60 in this disease. Female 10 40 On the other hand, macroscopic abnormalities such as Handedness ventricular enlargement, volume reductions of prefrontal Right 24 96 cortex and hippocampus and generalized brain reduction, Left 1 4 among many other features, are well-documented and Mean S.D. Age (years) 34.6 7.6 consistent findings (McCarley et al., 1999; Shenton et al., Education (years) 8.5 3.7 2001), although of relatively small effect (Heinrichs, Duration of illness (years) 15.7 9.3 2001). In addition, significant alterations in neuron size Age of onset of psychosis (years) 18.3 5.5 and morphology, as well as synaptic connectivity, have Positive and Negative Syndrome Scale score 55.7 19.9 also been reported in SZ (Harrison, 1999). Brief Psychiatric Rating Scale score 14.7 10.2 Negative Symptom Rating Scale score 13.9 9.5 The association of gene polymorphisms and brain structural or physiological abnormalities in SZ has been a S.D.: standard deviation. field of intense activity in recent years (Egan et al., 2001; Rujescu et al., 2002; Szeszko et al., 2003; Callicott et al., Gorham, 1962). Handedness was assessed with the 2005; Ho et al., 2005; Papiol et al., 2005; Prasad et al., Handedness Inventory (Briggs and Nebes, 1975). 2005; Szeszko et al., 2005), but a vast population of genes One experienced psychiatrist, blind to the MRI results, still needs to be evaluated for a better understanding of obtained all clinical ratings. A second set of samples, how the genetic alterations can influence brain morpho- derived from the same hospital, and consisting of 200 SZ genesis. All genes selected for our analyses, besides being and 200 controls was used for determining the allelic polymorphic, encode proteins important in SZ-related frequencies of the two single nucleotide polymorphisms processes such as neurogenesis, synaptogenesis, brain (SNPs) associated with the brain morphometric measure- symmetry, neuronal differentiation and migration, and ments analysis undertaken here. No MRI data were were preferentially mapped to genomic regions previous- available for these patients. More details about these ly associated with the disease. Associations of these genes patients and controls were given by Gregorio et al. (2006). and brain-structure differences were investigated in a set of 25 schizophrenia patients. This is the first study of 27 of 2.2. MRI acquisition and measurements these 32 polymorphisms and the first time that 24 of these 30 genes were evaluated in SZ. Structural MRI scans of the entire brain were obtained using a 1.5 T Philips Gyroscan S15-ACS scanner, with 2. Methods T1-FFE weighed continuous coronal slices (1.2 mm thick, FOV=240, matrix=256×256). Coronal images were 2.1. Patients oriented in planes perpendicular to the anterior-to- posterior commissural axis. Images were transferred to a Twenty-five schizophrenia patients were recruited at SUN Workstation, and measurements were manually the Institute of Psychiatry, Hospital das Clínicas, FMUSP, performed using the software Gyroview-HR 2.1, which São Paulo, Brazil. Diagnoses were made through also permitted reconstruction in axial and sagittal planes. structured interviews (SCIDP) based on DSM-IV criteria Regions of interest were chosen based on structures (First et al., 1996). Written informed consent was obtained frequently described in the literature as altered in SZ, from all participants after explanation of study protocols totaling 10 measures. Special focus was given to al- and purposes. The study was previously approved by the terations of ventricular, hippocampus and planum tem- ethics committee of the institution. Detailed demographic porale volumes, and to an index of cortical folding data of the patients are described in Table 1. Current (gyrification index). Details regarding anatomic land- symptom severity was measured using the Positive and marks and reliability analysis can be seen elsewhere Negative Syndrome Scale (PANSS) (Kay et al., 1987)and (Sallet et al., 2003a; Sallet et al., 2003b). An asymmetry the Brief Psychiatric Rating Scale (BPRS) (Overall and coefficient (AC) between hemispheres for the gyrification S.P. Gregório et al. / Psychiatry Research 165 (2009) 1–9 3 index was calculated using the algorithm: AC=[(Right− functional consequences, as most are non-synonymous Left)/0.5×(Right+Left)]×1000. Positive values indicate SNPs or SNPs located in putative promoter regions. rightward asymmetry; negative values indicate leftward Another three DNA polymorphisms (rs.7395206, asymmetry. rs.919001 and rs.861843), located in non-coding regions The ventricular–brain ratio (VBR) was obtained for of the genome, were used as control polymorphisms. each hemisphere using the algorithm: VBR=ventricular volume/brain volume×100. Measurements performed in 2.4. DNA extraction and genotyping the coronal plane: hippocampus and planum temporale were taken at every 1.2-mm continuous slice; and ventricles DNA was extracted from peripheral blood leukocytes and brain volumes at a distance of 3.6 mm. Volumes were of schizophrenic patients, according to salting-out meth- calculated multiplying the sum of areas by the thickness of ods for protein precipitation and DNA isolation (Laitinen slices. Absolute values were corrected using the algorithm: et al., 1994). Due to the diversity of the DNA corrected values=absolute value×brain volume/1000. polymorphisms investigated here, different approaches To ensure consistent delineation, all measurements were used for genotyping: 1) standard polymerase chain were taken by the same operator (P.C.S) while two other