Ventricular enlargement in schizophrenia is associated with a genetic polymorphism at the interleukin-1 receptor antagonist gene

Rapid Communication

www.elsevier.com/locate/ynimg NeuroImage 27 (2005) 1002 – 1006

Ventricular enlargement in schizophrenia is associated with a genetic polymorphism at the interleukin-1 receptor antagonist gene Sergi Papiol, a Vicente Molina, b Manuel Desco, c Araceli Rosa, a Santiago Reig, c Juan D. Gispert, c Javier Sanz, d Toma´s Palomo, d and Lourdes Fan˜ana´s a,* a

Unitat d’Antropologia, Departament de Biologia Animal, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain Department of Psychiatry, Hospital Clı´nico de Salamanca, Salamanca, Spain c Department of Experimental Medicine, Hospital General Universitario Gregorio Maran˜o´n, Madrid, Spain d Department of Psychiatry, Hospital Doce de Octubre, Edificio de Medicina Comunitaria, Madrid, Spain b

Received 10 January 2005; revised 9 April 2005; accepted 20 May 2005 Available online 12 July 2005 Magnetic resonance imaging (MRI) studies have shown some morphological and volumetric peculiarities in brains of schizophrenic patients. The authors explored the influence of genetic polymorphisms at interleukin-1B (IL-1B) and interleukin-1 receptor antagonist (IL-1RN) genes on these abnormalities. Hippocampus, lateral ventricles, and dorsolateral prefrontal cortex gray matter volumes were measured in a sample of 23 DSM-IV diagnosed schizophrenic patients of Spanish origin using MRI scans; MRI data were adjusted for age and brain volume using regression parameters from a healthy control group (n = 45). IL-1B and IL-1RN genes, involved in neurodevelopment and neurodegenerative processes, were analyzed in the patient sample. Patients carrying VNTR-allele*2 of IL-1RN gene showed a significant enlargement of both left ( P = 0.002) and right ( P = 0.01) ventricles. Sex and illness duration were controlled for in the analyses. Our results, though preliminary, suggest that IL-1RN gene might contribute to the ventricular volumetric changes observed in schizophrenic patients. D 2005 Elsevier Inc. All rights reserved. Keywords: Schizophrenia; IL-1RN; IL-1B; Genes; Ventricular enlargement; Endophenotype

Introduction Schizophrenia is a severe and chronic psychiatric disorder described in all cultures and populations, with worldwide lifetime prevalence of 1% (Jablensky et al., 1992). This mental disease is characterized by a broad range of symptoms affecting perception, language, emotion, reasoning, and motor activity. Although genetic contribution to schizophrenia has been widely documented (Cardno et al., 1999), it remains to be established which genes or which loci unequivocally contribute to the risk for this complex mental disorder. There are * Corresponding author. E-mail address: [email protected] (L. Fan˜ana´s). Available online on ScienceDirect (www.sciencedirect.com). 1053-8119/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2005.05.035

conflicting results from schizophrenia genetic studies based on categorical diagnoses. Heterogeneity of samples included under these categories as well as the variable psychopathological expression of the disease may account for some of these disappointing results. New approaches based on endophenotypes (or intermediate phenotypes), quantitative neurobiological traits related to genetic risk for schizophrenia which, hypothetically, involve simpler genetic pathways than whole clinical phenotypes, may shed light on such genetic studies (Egan and Weinberger, 1997). The most consistent findings in schizophrenia brain imaging studies (lateral and third ventricles, temporal lobe structures, orbitofrontal regions, subcortical structures, and cerebellum) should be potential endophenotypes of interest in this complex disorder. Among these changes, ventricular enlargement has been reported to be the most replicated result in schizophrenia neuroimaging studies (Chua and McKenna, 1995; Shenton et al., 2001; Suddath et al., 1990; Wright et al., 2000). Hence, several studies have focused on the genetic factors which might contribute to these abnormalities. In this vein, neuroimaging studies including healthy relatives of affected individuals, who are thought to have an increased genetic risk for schizophrenia, reported regional abnormalities which resemble those found in schizophrenic patients (Sharma et al., 1998; Staal et al., 2000). Otherwise, several twin studies pointed out the association between ventricle enlargement and psychosis in discordant monozygotic twin-pairs (Baare et al., 2001; Suddath et al., 1990), highlighting the role of environmental factors in the expression of this phenotype. The contribution of particular genes or specific genomic regions to brain morphological features both in healthy and schizophrenic subjects has yet to be ascertained, although several studies have suggested that genes such as neurotrophin-3, interleukin-1h, brainderived neurotrophic factor, tumor necrosis factor receptor-2, or prion protein gene would explain, at least in part, some brain morphological changes associated with schizophrenia (Kunugi et

S. Papiol et al. / NeuroImage 27 (2005) 1002 – 1006

al., 1999; Meisenzahl et al., 2001; Rujescu et al., 2002; Shihabuddin et al., 1996; Wassink et al., 1999, 2000). At this respect, an increasing amount of data emerging from neuroimaging (Meisenzahl et al., 2001) and genetic association studies (Katila et al., 1999; Kim et al., 2004; Papiol et al., 2004; Rosa et al., 2004) suggest that genetic variability located on interleukin-1 (IL-1) cluster might confer an increased risk to develop schizophrenia and contribute to some brain morphology variations observed in schizophrenic patients. IL-1B and IL-1RN genes, which encode for interleukin-1h (IL-1h) and interleukin-1 receptor antagonist (IL-1Ra), respectively, have been mapped to IL-1 cluster (chromosome 2q13). IL-1h (pro-inflammatory cytokine) and IL-1Ra (anti-inflammatory cytokine) are involved in neurodevelopmental processes (Nawa et al., 2000) as well as in acute and chronic neurodegeneration (Allan and Rothwell, 2001). Thus, all these data make genetic variability described in such genes suitable for analysis of their effect on brain morphology in schizophrenia. The aim of the present study was to investigate the relationship between genetic polymorphisms at the IL-1B and IL-1RN genes and morphological abnormalities previously reported in schizophrenia including (i) hippocampus (HIP), (ii) lateral ventricles (LV), and (iii) dorsolateral prefrontal cortex (DLPF) volumes. Methods Twenty-three DSM-IV schizophrenic patients were included in the present study (see Table 1 for demographic and clinical details of this sample). Eleven out of them were hospitalized inpatients. The sample consisted of ten first-episode and thirteen chronic patients. At the time of enrollment, all patients were treated with psychotropic medication. All subjects were of Caucasian Spanish origin and they provided written informed consent to participate in the study, which was approved by the hospital ethical committee. Exclusion criteria were: history of substance abuse, any other current comorbid Axis I diagnosis or psychoactive treatment, history of serious head trauma or any neurological or systemic disease with known effects on the central nervous system. Magnetic resonance imaging studies were acquired on a Philips Gyroscan 1.5-T scanner using a T1-weighted 3D gradient echo sequence with the following parameters: matrix size 256  256, pixel size 0.9  0.9 mm (FOV 256 mm), 100 slices 1.5 mm thick, flip angle 30-, repetition time 16 ms, echo time 4.6 ms. T2weighted sequences were also acquired for verification of CSF segmentations and for other clinical purposes (Turbo-Spin Echo, turbo factor 15, echo time 120 ms, matrix size 256  256, slice thickness 5.5 mm). In order to obtain volume measurements of the main brain lobes, we used a method for semi-automated segmentation of the Table 1 Socio-demographic and clinical characteristics of the sample Characteristics

Patients (n = 23)

Controls (n = 45)

Age: mean (SD) in years Sex: males/females Intracranial volume: mean (SD) in ml Illness duration: mean (SD) in years PANSS positive score: mean (SD) PANSS negative score: mean (SD)

34.0 (10.7) 14/9 1442.36 (133.0) 7.34 (6.25) 22.1 (7.0) 21.7 (8.1)

29.4 (9.0) 24/21 1401.6 (159.6)

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brain based on the Talairach proportional grid system, similar to those described in Andreasen et al. (1996) and Kates et al. (1999). Basically, we used a two-step procedure (Desco et al., 2001). The first step involved editing the MRI to remove skull and extracranial tissue, and an initial segmentation of cerebral tissues into gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF). In a second stage, we applied the Talairach reference system (Talairach and Tournoux, 1988) to define ROIs and to obtain volume data. MR images were processed using locally developed software that incorporates a variety of image processing and quantification tools (Desco et al., 2001). Segmentation of cerebral tissue was performed using an automated method included in the SPM2 (Statistical Parametric Mapping) program (Ashburner and Friston, 1997). The method performs a cluster analysis with modified mixture model and a priori information about the likelihoods of each MRI voxel being one of 4 tissue types: GM, WM, CSF, and ‘‘other tissues.’’ The a priori information is anatomical templates that represent an Faverage_ brain and provide information about the spatial distribution of the different brain tissues. The algorithm also removes the effect of radiofrequency field inhomogeneities (Ashburner and Friston, 2000). This segmentation was checked for inconsistencies and manually corrected whenever necessary by an experienced radiologist blinded to the diagnosis. In the second stage, the edited MRI (without extracranial tissue) was used to build the Talairach grid, and the ROIs were obtained by superimposing the 3D tissue masks corresponding to WM, GM, and CSF onto each subject’s Talairach reference grid, where the regions of interest were defined as sets of cells. On this MRI with the Talairach grid, volume for each tissue type was measured by totaling the data from the grid cells associated with each ROI (Desco et al., 2001). ROI variables included in the analysis were dorsolateral prefrontal cortex (DLPF), lateral ventricles (LV), and hippocampus (HIP). The DLPF was defined as the Talairach grid cells of the GM tissue encompassing Brodmann’s areas 8, 9, 10, and 46. Hippocampal volume measurements were obtained considering only the GM tissue contained within relevant cells (E2b10 and E3b10) described in the Talairach atlas (Talairach and Tournoux, 1988). The lateral ventricles were measured as the CSF tissue in the Talairach grid cells encompassing this region (Kates et al., 1999; Swayze et al., 1996). The validity of the Talairach-based procedure as a suitable automated segmentation tool in schizophrenia research has been previously proven (Andreasen et al., 1996; Ho et al., 2003; Kates et al., 1999). In our implementation, all manual procedures were performed by a single operator, thus avoiding any potential interrater variability. Reliability of the method was assessed by repeating the whole segmentation procedure in a sample of 5 cases randomly selected. Values of ICC ranged from 0.96 to 0.99 for regional GM measurements, and from 0.89 to 0.99 for CSF data. Since age and total cranial size are known factors affecting regional cerebral volumes, their effect was removed by using the residuals from the regression models obtained from a group of 45 healthy individuals (see Table 1 for details), following the procedure of Pfefferbaum et al. (1992). After this correction, volume variables become expressed as deviations from the expected volumes of healthy individuals of the same age as the patient. Thus, negative residuals represent a quantitative measurement of atrophy.

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Table 2 Allelic and genotypic frequencies of

511C/T and 86-bp VNTR polymorphisms of IL-1B and IL-1RN genes in schizophrenic patients

Genotypic frequencies IL-1B 511 C/T N 23 IL-1RN VNTR N 23

Allelic frequencies

allele*1/allele*1 10 (43.5%)

allele*1/allele*2 7 (30.4%)

allele*2/allele*2 6 (26.1%)

allele*1/allele*1 10 (43.5%)

allele*1/allele*2 11 (47.8%)

allele*2/allele*2 1 (4.3%)

Mean comparison (Student’s t-test) analyses were conducted in order to detect the effect of genotype on the volume of HIP, LV, and DLPF. Genomic DNA was extracted from blood samples using standard phenol-chloroform methods. A 511C/T AvaI polymorphic site (rs16944) located on the promoter region of IL-1B gene and an 86-bp repeat of IL-1RN gene were genotyped as described previously by Katila et al. (1999). Briefly, allele 1 ( 511 C) of IL1B gene completes an AvaI restriction site, while allele 2 ( 511 T) gives an intact product. When the variable nucleotide tandem repeat (VNTR) in intron 2 of IL-1RN gene was analyzed, we detected the three commonest alleles of this polymorphism (A1, A2, and A3) which correspond to four, two, and five repeats, respectively.

Results Frequencies observed for genotypes and alleles of the genetic polymorphisms in our sample of schizophrenic patients are shown in Table 2. Our sample showed Hardy – Weinberg equilibrium for the genotypic frequencies of IL-1B promoter polymorphism and IL-1RN intronic VNTR (data not shown). Two subgroups were generated by placing allele*2 carriers (A1/ A2 and A2/A2) of VNTR of IL-1RN gene into one group (n = 12) and putting together the rest of the no-carriers genotypes (A1/A1 and A1/A3) into the other (n = 11). There were no differences in

allele*1/allele*3 1 (4.3%)

allele*1 27 (58.7%)

allele*2 19 (41.3%)

allele*1 32 (69.6%)

allele*2 13 (28.3%)

allele*3 1 (2.1%)

terms of illness duration, sex distribution, and treatment (typical/ atypical) between both subgroups (data not shown). After correction for age and brain volume using regression parameters from healthy control group, patients who were carriers of VNTR allele*2 showed a significant enlargement of both left (t = 3.504, df = 21, P = 0.002) and right (t = 2.784; df = 21, P = 0.01) ventricles with respect to those who were no-carriers (see Fig. 1). When the same analysis was conducted with respect to hippocampus and dorsolateral prefrontal cortex, we did not find differences for the residual volumes according to IL-1RN genotype in either side (HIP: t < 1.6, df = 21, P = ns; DLPF: t < 1. 5, df = 21, P = ns). For the analysis of IL-1B polymorphism, we took into account a previous neuroimaging study (Meisenzahl et al., 2001) and we collapsed in a single group allele 2 ( 511 T) carriers (allele2/ allele2 and allele1/allele2) for comparisons with allele 1 homozygous. Results showed that this polymorphism has no effect on the volumes of lateral ventricles (t > 1.7, df = 21, P = ns), hippocampus (t > 0.2, df = 21, P = ns), and dorsolateral prefrontal cortex (t < 0.7, df = 21, P = ns) in either side. Since the sample was heterogeneous in terms of illness duration, we checked the significance of volume differences of the regions of interest in first episode vs. chronic patients using Student’s t-test for independent samples. No significant differences between these groups were detected for the residual volumes of hippocampus and ventricles in either side (t < 0.9, df = 21, P = ns in all cases). However, right prefrontal residuals were significantly

Fig. 1. Scatterplots of left and right ventricle volumes after correction for age and brain volume by using regression parameters from healthy control group. These residuals, in the y axis, represent a quantitative measure of ventricular dilation (if positive) with respect to a control sample (see Methods). Patients who were allele*2 carriers of IL-1RN gene polymorphism show a significant increase in both left ( P = 0.002) and right ( P = 0.01) cerebral ventricles.

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lower in chronic patients (mean 3.6, SD 2.9) as compared to first episodes (mean 0.4, SD 2.9; t = 2.33, df = 21, P = 0.03).

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In conclusion, the present finding enhances the interest of MRIbased intermediate phenotypes in ongoing and future genetic studies of schizophrenia.

Discussion Acknowledgments Though preliminary, our results suggest that variability at the IL-1RN gene might contribute to explain the ventricular volumetric variation observed in schizophrenic patients and, therefore, they reinforce the validity of this hypothetical intermediate phenotype in schizophrenia research. Our data are consistent with a recent study showing that a haplotypic combination within IL-1 cluster which includes allele*2 of IL-1RN gene confers an increased risk (2.5 times) to develop schizophrenia (Papiol et al., 2004). Likewise, another recent study has detected the effect of the same allelic variant per se, conferring a 2.24 times increased risk for the disease (Kim et al., 2004). In addition, a previous neuroimaging study found an association of allele 2 ( 511 T) of IL-1B gene promoter polymorphism with bifrontal – temporal gray matter volume deficits and generalized white matter tissue deficits in schizophrenic patients (Meisenzahl et al., 2001). However, further analyses are necessary in order to clarify the role of IL-1 cluster genetic variability in brain morphology, its relationship with structural and volumetric abnormalities observed in brains of schizophrenic patients, and its significance in the definition of endophenotypes based on neuroimage (Egan and Weinberger, 1997). At this point, it is interesting to note a recent in vitro study showing that pro-inflammatory cytokines (IL-1h, IL-6, and TNFa) decrease dendritic development of cortical neurons (Gilmore et al., 2004). Unfortunately, little is known about processes which may link ventricular enlargement to schizophrenia symptoms. In the mammalian developing brain, neurons and macroglia arise and migrate from precursors in the germinal layers (ventricular and subventricular zones), which are surrounding ventricles (Doetsch et al., 1999). Hypothetically, ventricular enlargement may reflect subtle neuropathologic processes in these zones (as, for example, abnormal neuronal migration) with dramatic effects in cerebral regions related to illness. At this respect, a recent study has demonstrated that enlarged ventricles in schizophrenic patients are associated with regionally specific reductions, especially in thalamic candidate regions adjacent to the body of the ventricles (Gaser et al., 2004). Concerning the implications of this study for understanding the neuropathology of schizophrenia, from our results it could be hypothesized that genetic variability located on IL-1RN gene and/ or other genes also involved in neurodevelopment is affecting brain morphology, leading to subtle variations such as ventricular enlargement. Indeed, heritability estimates for lateral ventricular volumes have shown values up to 85% (Reveley et al., 1984). Furthermore, a recent study has pointed out the great influence of genetics on lateral ventricle shape (Styner et al., 2005). Such a genetic implication in morphologic brain changes in schizophrenia is not necessarily opposed to a role for environmental factors. In fact, subtle abnormalities in certain brain regions might define genetically vulnerable individuals in front of environmental agents such as treatments, chronicity, intrauterine infections, and obstetric problems (van Haren et al., 2004). Further studies in larger samples would be necessary in order to rule out the possibility of a spurious association in our results.

The authors would like to thank the participating patients and their families, whose generous contributions have made this study possible. We are grateful to Fernando Sarramea and Rogelio Luque for their help in sample collection. Sergi Papiol was supported by a grant of the Ministry of Education and Culture of Spain. This study was supported by grants from Fundacio´ ‘‘La Caixa’’ (99-111-00), (99-042-00), and FIS (02/3095) and IM3 (G03/185).

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