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No evidence for association of the dysbindin gene [DTNBP1] with schizophrenia in an Irish population-based study
Schizophrenia Research 60 (2003) 167 – 172 www.elsevier.com/locate/schres
No evidence for association of the dysbindin gene [DTNBP1] with schizophrenia in an Irish population-based study Derek W. Morris a,*, Kevin A. McGhee a, Siobhan Schwaiger a, Paul Scully b, John Quinn b, David Meagher b, John L. Waddington b,c, Michael Gill a, Aiden P. Corvin a a
Neuropsychiatric Genetics Group, Department of Genetics, Trinity College, Dublin 2, Ireland b Stanley Research Unit, St. Davnet’s Hospital, Monaghan, Ireland c Department of Clinical Pharmacology, Royal College of Surgeons, 123 St. Stephen’s Green, Dublin 2, Ireland Received 28 October 2002; accepted 13 December 2002
Abstract A recent family-based association study identified a putative association between variants in the dystrobrevin binding protein 1 (dysbindin) gene (DTNBP1) and schizophrenia. This study used a sample of 270 Irish pedigrees multiply affected with schizophrenia. We attempted to replicate these findings in an independent Irish sample of 219 schizophrenia cases and 231 controls. No evidence was found to suggest an association between the DTNBP1 gene and schizophrenia in our sample. Possible reasons for these findings are discussed. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Schizophrenia; Dysbindin; DTNBP1; Association
1. Introduction Schizophrenia (MIM 181500; http://www.ncbi. nlm.nih.gov/Omim/) is a common psychiatric disorder with a lifetime prevalence of f 1%, which presents with psychotic symptoms (delusions and hallucinations), thought disorder and deficit features described as ‘negative’ symptoms. Although schizophrenia is highly heritable, the genetic etiology is complex and identifying replicable susceptibility loci, let alone * Corresponding author. Tel.: +353-1-608-2444; fax: +353-1679-8558. E-mail address: [email protected] (D.W. Morris).
genes, has proved to be problematic. However, susceptibility genes for complex disorders are increasingly being identified (Hugot et al., 2001; Hakonarson et al., 2002). Using a sample of 270 Irish families multiply affected with schizophrenia, Straub et al. (2002a) identified a multipoint linkage peak at 6p22 bordered by the markers D6S260 and D6S1676. This finding has received support from a number of other studies (Schwab et al., 1995; Hwu et al., 2000) as well as the Schizophrenia Linkage Collaborative Group for Chromosomes 3, 6 and 8 (1996). In addition, a recent meta-analysis of all completed schizophrenia genome scans suggested the presence of several susceptibility loci, including the chromosome 6p locus (Levinson et
0920-9964/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0920-9964(02)00527-3
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al., 2002). Investigating this linkage further in the same sample, Straub et al. (2002b) reported a positive genetic association between schizophrenia and variants within the dystrobrevin binding protein 1 (dysbindin) gene (DTNBP1). The DTNBP1 gene contains 10 exons and spans a region of 140 kb on 6p22.3 (Ensembl gene ID: ENSG00000047579; http://www.ensembl.org/). DTNBP1 is predicted to produce, by alternative splicing, nine different transcripts altogether encoding nine proteins (enter DTNBP1 in AceView; http:// www.ncbi.nlm.nih.gov/IEB/Research/Acembly/). The protein dysbindin is found in a small subset of axons with large synaptic terminals, such as mossy fibres in the cerebellum, hippocampus and cochlear nuclei (Benson et al., 2001). Some of these axons are localized to anatomical regions implicated in schizophrenia (McCarley et al., 1999), and Straub et al. (2002b) speculate on the possible role of dysbindin in the pathogenesis of schizophrenia through modulation of synaptic signalling and plasticity. This finding of association in families multiply affected with schizophrenia is important but it is necessary to establish if this association exists in a sample of patients with schizophrenia who were not selected for familiality. We have performed association analyses using SNPs from the DTNBP1 gene in an independent sample of Irish schizophrenia cases and controls.
included in this investigation met criteria for schizophrenia or schizo-affective disorder. This is equivalent to the D1 – D2 ‘narrow’ diagnostic category used by Straub et al. (2002b). Family history of a diagnosis of schizophrenia or other psychotic disorder was obtained from the proband and was confirmed where possible with a family member, medical or nursing staff or case notes. The control sample, drawn from Irish blood donors, was not specifically screened for psychiatric illness; however, donors were not taking regular prescribed medication as such individuals are excluded from blood donation in Ireland. 2.2. SNP identification The SNPs genotyped in this study are detailed in Table 1. Five of these SNPs (A, B, C, E and F) were identified by genotyping SNPs taken from the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) in a pooled DNA sample of 100 individuals using SNaPshotk (Applied Biosystems). The remaining three SNPs (D, G and H) were taken from Straub et al. (2002b). The positioning of these SNPs within the DTNBP1 gene can be seen in Fig. 1. Included among the SNPs typed are five SNPs that were genotyped in the Straub study, three of which provided the strongest evidence of association in that study. 2.3. SNP genotyping
2. Materials and methods 2.1. Sample collection and diagnostic assessment The sample consisted of 219 cases and 231 controls from the Republic of Ireland. Ethics Committee approval for the study was obtained from all participating hospitals and centres. All cases gave written informed consent and were interviewed by a psychiatrist or psychiatric nurse trained to use the Structured Clinical Interview for DSM (SCID). Diagnosis was based on DSM-IIIR criteria using all available information (interview, family or staff report and chart review). All cases were over 18 years of age, of Irish origin and had been screened to exclude substance-induced psychotic disorder or psychosis due to a general medical condition. Cases
All SNPs were genotyped using the SNaPshotk method of single base extension (Applied Biosystems) that employs the principle of extending an unlabelled oligonucleotide primer in the presence of fluorescently labelled ddNTPs. Each of the four ddNTPs is tagged by a different fluorescent dye. Hence, when the primer extension products are run on an ABI DNA Sequencer or ABI Genetic Analyser (in this case an ABI PRISM 377 DNA Sequencer), the specific allele products can be differentiated from each other on the basis of which fluorescent dye they carry. This method can be used for both individual and pooled genotyping (Pastinen et al., 1996; Norton et al., 2002). Details of PCR primers and PCR reaction conditions and extension primers and extension reaction conditions for all SNPs are available on request.
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Table 1 SNPs from the DTNBP1 gene
A B C D E F G H
dbSNP IDa
Alternative marker nameb
Polymorphism
Frequencyc in controls
Frequencyc in casesd
p valuee
Frequencyc in casesf
p valueg
rs1047631 rs760666 rs2619539 rs3213207 rs2619542 rs2619550 rs2005976 rs760761
– P1287 P1655 P1635 – – P1757 P1320
A/G C/T G/C G/A C/T C/G A/G C/T
0.840 0.773 0.485 0.115 0.524 0.195 0.199 0.784
0.868 0.759 0.486 0.100 0.523 0.163 0.162 0.826
0.277 0.632 0.964 0.490 0.980 0.212 0.149 0.105
0.841 0.769 0.438 0.121 0.569 0.212 0.205 0.788
0.975 0.933 0.352 0.838 0.359 0.660 0.890 0.916
a
NCBI SNP Cluster ID, from the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/). Marker names used by Straub et al. for those SNPs genotyped in that study. c Of first allele shown. d All cases. e p value calculated for all cases and all controls. f Allele frequency for only those cases that are family history positive (n = 65; see Materials and methods). g p value calculated for cases that were family history positive and all controls. b
2.4. Statistical analysis
3. Results
SNPs were tested for association with the phenotype by using a 2 2 contingency table to calculate a v2 statistic. Haplotype analysis was performed using the HAPMAX programme (http://www.uwcm.ac.uk/ uwcm/mg/download). HAPMAX was first used to estimate marker – marker haplotype frequencies on transmitted and nontransmitted chromosomes in cystic fibrosis families (Krawczak et al., 1988). HAPMAX employs the EM algorithm to calculate haplotype frequencies. Intermarker linkage disequilibrium (LD) was measured using DV (Lewontin, 1964). DV values were calculated using the GOLD programme (http://www.sph.umich.edu/csg/abecasis/ GOLD/).
Of the 219 cases, 176 were diagnosed as schizophrenia and 43 as schizo-affective disorder. The frequencies of each SNP in the case and control groups are detailed in Table 1 along with the p value for each test of association. All SNPs were found to be in Hardy –Weinberg equilibrium in both the case and control samples. An exception to this was SNP A, which was not in Hardy –Weinberg equilibrium in either the cases or the controls ( p = 0.024 and p = 0.018, respectively). This is likely to be due to the low frequency of the minor allele in both samples. A comparison of the allele frequencies of the five SNPs (B, C, D, G and H in Table 1) genotyped in both studies shows that all five SNPs have strikingly
Fig. 1. Map of DTNBP1 showing the physical positioning of the eight SNPs (detailed in Table 1) genotyped in this study within the exonic structure of the gene. The intervals between adjacent SNPs are given in base pairs (bp).
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similar allele frequencies. In our study, the minor allele frequencies of SNPs B, C, D, G and H in the combined sample of cases and controls are 0.234, 0.485, 0.108, 0.181 and 0.196, respectively (separate frequencies for cases and controls are in Table 1). These compare to frequencies of 0.246, 0.463, 0.102, 0.167 and 0.178 in the total Straub sample (calculated using TRANSMIT; Straub et al., 2002b). None of the SNPs was found to be associated with illness. Of the five SNPs that were genotyped by Straub et al. in their sample, three of them (D, G and H) were found to be associated with schizophrenia when the sample was limited to one affected offspring per family under the ‘‘narrow’’ diagnostic category (Straub et al., 2002b). Haplotype analyses were performed with haplotypes constructed from adjacent markers in a sliding window fashion across the gene. HAPMAX was used to calculate haplotype frequencies for two-, three- and four-marker haplotypes. Association analyses were performed using the two-, three- and four-marker haplotypes but no evidence of association was found (data not shown). DVwas calculated for all pairs of SNPs in both the cases and the controls. These data are presented in Tables 2 and 3. The pattern of LD is broadly similar in both samples. SNPs B and D are both in very tight LD with all other SNPs across the gene. SNP A, the most telomeric SNP, is in moderate LD with the remaining SNPs. After that, there is a clear divide in the pattern of LD. There is strong LD between SNPs C and E, and between SNPs F, G and H, but there is only weak LD between these two sets of SNPs. Straub et al. (2002b) also reported pairwise DVvalues for all SNPs genotyped in their study. For SNPs that have been genotyped in both studies, DVvalues are very similar. For example, in our control sample, the DV values Table 2 DVvalues calculated for all pairs of SNPs (A – H) in the case sample
A B C D E F G H
A :::
B
1.00 0.62 0.92 0.54 0.62 0.62 0.63
::: 0.97 1.00 1.00 1.00 1.00 1.00
C
D
E
F
G
H
::: 1.00 1.00 0.26 0.23 0.23
::: 0.93 0.88 0.88 1.00
::: 0.24 0.21 0.21
::: 0.98 0.98
::: 1.00
:::
Table 3 DVvalues calculated for all pairs of SNPs (A – H) in the control sample
A B C D E F G H
A :::
B
0.99 0.35 0.86 0.30 0.50 0.50 0.49
::: 0.97 0.99 1.00 1.00 1.00 1.00
C
D
E
F
G
H
::: 1.00 1.00 0.02 0.03 0.02
::: 1.00 0.90 0.90 1.00
::: 0.03 0.01 0.03
::: 1.00 1.00
::: 1.00
:::
calculated for the SNP pairs B and C, C and G, and D and H are 0.97, 0.23 and 1.00, respectively. These correspond to values of 1.00, 0.15 and 0.93 in Straub et al. (2002b).
3. Discussion There are striking similarities in both the allele frequencies and DV values calculated for the SNPs genotyped both in this study and in that of Straub et al. (2002b). These similarities suggest that the samples used in both studies have been drawn from the same homogeneous population. Therefore, it is surprising that we do not identify the association reported by Straub et al. (2002b). There are a number of possible reasons for this. The first relates to known differences between the samples. Our sample was composed of individuals with schizophrenia drawn from the Irish population, whereas in the study of Straub et al. (2002b), cases were derived from pedigrees with multiply affected individuals. The genetic architecture of schizophrenia in such families may differ from that of unselected cases. Multiply affected families may represent a distinct genetic subset of schizophrenia. Earlier studies of schizophrenia based on a sporadic/familial distinction have been criticized as oversimplistic (Kendler, 1988). However, the assumption that most cases of schizophrenia share similar genetic aetiology may be equally simplistic. Such considerations may be relevant to the finding of association in Straub’s family-based sample and the absence of association in the present population-based sample. To examine this, we stratified our sample of cases into those that had a first or second degree
D.W. Morris et al. / Schizophrenia Research 60 (2003) 167–172
relative with schizophrenia or schizo-affective disorder and those that did not. Association analysis was then performed in the SNP data using only those cases that were ‘family history positive’. Single marker analysis (see Table 1) and haplotype analysis (data not presented) provided no evidence of association and hence does not support the sporadic/familial divide. However, given the small sample size of the family history positive cases (n = 65), we cannot make any firm conclusions on this. A second possibility is that our study lacks power. Because the study of Straub et al. (2002b) does not include odds ratios/relative risk information for the associated SNPs D, G and H, we are unable to directly calculate the power of our sample to replicate their findings. However, we can address this issue indirectly by calculating odds ratios (OR) with 95% confidence intervals (95% CI) for SNPs D, G and H in our sample. These data are presented for both our full case-control sample and for the subset of family history positive cases in Table 4. Examining these data allows us to make certain post hoc observations. Firstly, from the full schizophrenia sample, we can exclude, with 95% certainty, an OR>1.34 for association with SNPs D, G or H. Secondly, examining the family history positive cases indicates that we can exclude, with 95% certainty, an OR>2 for the same SNPs. A third possibility relates to the differences in analytical methods employed. The findings of the Straub study are based on transmission distortion in data from nuclear families. The TDT test identifies linkage in the presence of association (Spielman et al., 1993), a finding that assumes the independence of the nuclear families. If this assumption is invalid and an unrecognised pedigree structure exists in a subset of the Straub et al. (1995) nuclear families, the reported
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association finding may reflect the previously reported linkage. This is probably unlikely given the large numbers of families recruited from throughout Ireland but would explain our failure to replicate the association. Alternatively, given that the findings of Straub et al. (2002b) are family-based, they may represent segregation distortion in this chromosomal region and hence evidence of association would not be found in our case-control sample. Finally, is it possible that we have missed the association? We have not genotyped every SNP from the Straub study in our sample. However, we have tested the three SNPs that provided the strongest evidence of association in that study. In addition, we have genotyped five other SNPs from across the gene. Single marker analysis of these SNPs does not suggest an association between variants in the DTNBP1 gene and schizophrenia in our sample. We performed haplotype analysis with haplotypes constructed from adjacent SNPs. These haplotypes span the region of strongest association as adjudged by haplotype analysis in the Straub study. Again, we found no evidence of association in our sample; hence, this explanation is possible but unlikely. Further studies on this and other samples will be required to determine which of the above explanations is most likely to establish the validity of the putative association between DTNBP1 and schizophrenia. In particular, we are unable to exclude the possibility that the DTNBP1 association is specific to a familial subtype of schizophrenia identified in these multiply affected pedigrees. This possibility must be tested in additional familial and population-based samples. Clarification of this issue could profoundly effect future study design and our understanding of the aetiology of schizophrenia.
Acknowledgements Table 4 Odds ratios with 95% confidence intervals from association study data SNP
D G H
Schizophrenia cases/controls
Family history positive cases/controls
OR
95% CI
OR
95% CI
0.86 0.78 0.76
0.55 – 1.34 0.54 – 1.11 0.54 – 1.07
1.06 1.03 0.97
0.56 – 2.00 0.62 – 1.71 0.59 – 1.60
The authors gratefully acknowledge the contribution of all participating individuals and institutions in this study. Dr. Morris is supported by the Higher Education Authority. Dr. Corvin is a Wellcome Trust Research Fellow in Mental Health. The authors would like to thank the Health Research Board (Ireland) and the Stanley Medical Research Institute (USA) for their support of this research.
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References Benson, M.A., Newey, S.E., Martin-Rendon, E., Hawkes, R., Blake, D.J., 2001. Dysbindin, a novel coiled-coil-containing protein that interacts with the dystrobrevins in muscle and brain. J. Biol. Chem. 276, 24232 – 24241. Hakonarson, H., Bjornsdottir, U.S., Halapi, E., Palsson, S., Adalsteinsdottir, E., Gislason, D., Finnbogason, G., Gislason, T., Kristjansson, K., Arnason, T., Birkisson, I., Frigge, M.L., Kong, A., Gulcher, J.R., Stefansson, K., 2002. A major susceptibility gene for asthma maps to chromosome 14q24. Am. J. Hum. Genet. 71, 483 – 491. Hugot, J., Chamaillard, M., Zouali, H., Lesage, S., Cezard, J., Belaiche, J., Almer, S., Tysk, C., O’Morain, C.A., Gassull, M., Binder, V., Finkel, Y., Cortot, A., Modigliani, R., Laurent-Puig, P., Gower-Rousseau, C., Macry, J., Colombel, J., Sahbatou, M., Thomas, G., 2001. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 411, 599 – 603. Hwu, H.G., Lin, M.W., Lee, P.C., Lee, S.F., Ou-Yang, W.C., Liu, C.M., 2000. Evaluation of linkage of markers on chromosome 6p with schizophrenia in Taiwanese families. Am. J. Med. Genet. 96, 74 – 78. Kendler, K.S., 1988. The sporadic v. familial classification given aetiological heterogeneity: II. Power analyses. Psychol. Med. 18, 991 – 999. Krawczak, M., Konecki, D.S., Schmidtke, J., Duck, M., Engel, W., Nutzenadel, W., Trefz, F.K., 1988. Allelic association of the cystic fibrosis locus and two DNA markers, XV2c and KM19, in 55 German families. Hum. Genet. 80, 78 – 80. Levinson, D.F., Lewis, C.M., Wise, L.H., et al., 2002. Meta-analysis of genome scans for schizophrenia. Am. J. Med. Genet. 114 (SL2), 697 – 890. Lewontin, R.C., 1964. The interaction of selection and linkage I general considerations: heterotic models. Genetics 49, 49 – 67. McCarley, R.W., Wible, C.G., Frumin, M., Hirayasu, Y., Levitt, J.J., Fischer, I.A., Shenton, M.E., 1999. MRI anatomy of schizophrenia. Biol. Psychiatry 45, 1099 – 1119. Norton, N., Williams, N.M., Williams, H.J., Spurlock, G., Kirov,
G., Morris, D.W., Hoogendoorn, B., Owen, M.J., O’Donovan, M.C., 2002. Universal, robust, highly quantitative SNP allele frequency measurement in DNA pools. Hum. Genet. 110, 471 – 478. Pastinen, T., Partanen, J., Syvanen, A.C., 1996. Multiplex, fluorescent, solid phase minisequencing for efficient screening of DNA sequence variation. Clin. Chem. 42, 1391 – 1397. Schizophrenia Linkage Collaborative Group for Chromosomes 3, 6 and 8, 1996. Additional support for schizophrenia linkage on chromosomes 6 and 8: a multicenter study. Am. J. Med. Genet. 67, 580 – 594. Schwab, S.G., Albus, M., Hallmayer, J., Honig, S., Borrmann, M., Lichtermann, D., Ebstein, R.P., Ackenheil, M., Lerer, B., Risch, N., Maier, W., Wildenauer, D.B., 1995. Evaluation of a susceptibility gene for schizophrenia on chromosome 6p by multipoint affected sib-pair linkage analysis. Nat. Genet. 11, 325 – 327. Spielman, R.S., McGinnis, R.E., Ewens, W.J., 1993. Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am. J. Hum. Genet. 52, 506 – 516. Straub, R.E., MacLean, C.J., O’Neill, F.A., Burke, J., Murphy, B., Duke, F., Shinkwin, R., Webb, B.T., Zhang, J., Walsh, D., Kendler, K.S., 1995. A potential vulnerability locus for schizophrenia on chromosome 6p24-22: evidence for genetic heterogeneity. Nat. Genet. 11, 287 – 293. Straub, R.E., MacLean, C.J., Ma, Y., Webb, B.T., Myakishev, M.V., Harris-Kerr, C., Wormley, B., Sadek, H., Kadambi, B., O’Neill, F.A., Walsh, D., Kendler, K.S., 2002a. Genome-wide scans of three independent sets of 90 Irish multiplex schizophrenia families and follow-up of selected regions in all families provides evidence for multiple susceptibility genes. Mol. Psychiatry 7, 542 – 559. Straub, R.E., Jiang, Y., MacLean, C.J., Ma, Y., Webb, B.T., Myakishev, M.V., Harris-Kerr, C., Wormley, B., Sadek, H., Kadambi, B., Cesare, A.J., Gibberman, A., Wang, X., O’Neill, F.A., Walsh, D., Kendler, K.S., 2002b. Genetic variation in the 6p22.3 gene DTNBP1, the human ortholog of the mouse dysbindin gene, is associated with schizophrenia. Am. J. Hum. Genet. 71, 337 – 348.
No evidence for association of the dysbindin gene [DTNBP1] with schizophrenia in an Irish population-based study Derek W. Morris a,*, Kevin A. McGhee a, Siobhan Schwaiger a, Paul Scully b, John Quinn b, David Meagher b, John L. Waddington b,c, Michael Gill a, Aiden P. Corvin a a
Neuropsychiatric Genetics Group, Department of Genetics, Trinity College, Dublin 2, Ireland b Stanley Research Unit, St. Davnet’s Hospital, Monaghan, Ireland c Department of Clinical Pharmacology, Royal College of Surgeons, 123 St. Stephen’s Green, Dublin 2, Ireland Received 28 October 2002; accepted 13 December 2002
Abstract A recent family-based association study identified a putative association between variants in the dystrobrevin binding protein 1 (dysbindin) gene (DTNBP1) and schizophrenia. This study used a sample of 270 Irish pedigrees multiply affected with schizophrenia. We attempted to replicate these findings in an independent Irish sample of 219 schizophrenia cases and 231 controls. No evidence was found to suggest an association between the DTNBP1 gene and schizophrenia in our sample. Possible reasons for these findings are discussed. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Schizophrenia; Dysbindin; DTNBP1; Association
1. Introduction Schizophrenia (MIM 181500; http://www.ncbi. nlm.nih.gov/Omim/) is a common psychiatric disorder with a lifetime prevalence of f 1%, which presents with psychotic symptoms (delusions and hallucinations), thought disorder and deficit features described as ‘negative’ symptoms. Although schizophrenia is highly heritable, the genetic etiology is complex and identifying replicable susceptibility loci, let alone * Corresponding author. Tel.: +353-1-608-2444; fax: +353-1679-8558. E-mail address: [email protected] (D.W. Morris).
genes, has proved to be problematic. However, susceptibility genes for complex disorders are increasingly being identified (Hugot et al., 2001; Hakonarson et al., 2002). Using a sample of 270 Irish families multiply affected with schizophrenia, Straub et al. (2002a) identified a multipoint linkage peak at 6p22 bordered by the markers D6S260 and D6S1676. This finding has received support from a number of other studies (Schwab et al., 1995; Hwu et al., 2000) as well as the Schizophrenia Linkage Collaborative Group for Chromosomes 3, 6 and 8 (1996). In addition, a recent meta-analysis of all completed schizophrenia genome scans suggested the presence of several susceptibility loci, including the chromosome 6p locus (Levinson et
0920-9964/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0920-9964(02)00527-3
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al., 2002). Investigating this linkage further in the same sample, Straub et al. (2002b) reported a positive genetic association between schizophrenia and variants within the dystrobrevin binding protein 1 (dysbindin) gene (DTNBP1). The DTNBP1 gene contains 10 exons and spans a region of 140 kb on 6p22.3 (Ensembl gene ID: ENSG00000047579; http://www.ensembl.org/). DTNBP1 is predicted to produce, by alternative splicing, nine different transcripts altogether encoding nine proteins (enter DTNBP1 in AceView; http:// www.ncbi.nlm.nih.gov/IEB/Research/Acembly/). The protein dysbindin is found in a small subset of axons with large synaptic terminals, such as mossy fibres in the cerebellum, hippocampus and cochlear nuclei (Benson et al., 2001). Some of these axons are localized to anatomical regions implicated in schizophrenia (McCarley et al., 1999), and Straub et al. (2002b) speculate on the possible role of dysbindin in the pathogenesis of schizophrenia through modulation of synaptic signalling and plasticity. This finding of association in families multiply affected with schizophrenia is important but it is necessary to establish if this association exists in a sample of patients with schizophrenia who were not selected for familiality. We have performed association analyses using SNPs from the DTNBP1 gene in an independent sample of Irish schizophrenia cases and controls.
included in this investigation met criteria for schizophrenia or schizo-affective disorder. This is equivalent to the D1 – D2 ‘narrow’ diagnostic category used by Straub et al. (2002b). Family history of a diagnosis of schizophrenia or other psychotic disorder was obtained from the proband and was confirmed where possible with a family member, medical or nursing staff or case notes. The control sample, drawn from Irish blood donors, was not specifically screened for psychiatric illness; however, donors were not taking regular prescribed medication as such individuals are excluded from blood donation in Ireland. 2.2. SNP identification The SNPs genotyped in this study are detailed in Table 1. Five of these SNPs (A, B, C, E and F) were identified by genotyping SNPs taken from the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) in a pooled DNA sample of 100 individuals using SNaPshotk (Applied Biosystems). The remaining three SNPs (D, G and H) were taken from Straub et al. (2002b). The positioning of these SNPs within the DTNBP1 gene can be seen in Fig. 1. Included among the SNPs typed are five SNPs that were genotyped in the Straub study, three of which provided the strongest evidence of association in that study. 2.3. SNP genotyping
2. Materials and methods 2.1. Sample collection and diagnostic assessment The sample consisted of 219 cases and 231 controls from the Republic of Ireland. Ethics Committee approval for the study was obtained from all participating hospitals and centres. All cases gave written informed consent and were interviewed by a psychiatrist or psychiatric nurse trained to use the Structured Clinical Interview for DSM (SCID). Diagnosis was based on DSM-IIIR criteria using all available information (interview, family or staff report and chart review). All cases were over 18 years of age, of Irish origin and had been screened to exclude substance-induced psychotic disorder or psychosis due to a general medical condition. Cases
All SNPs were genotyped using the SNaPshotk method of single base extension (Applied Biosystems) that employs the principle of extending an unlabelled oligonucleotide primer in the presence of fluorescently labelled ddNTPs. Each of the four ddNTPs is tagged by a different fluorescent dye. Hence, when the primer extension products are run on an ABI DNA Sequencer or ABI Genetic Analyser (in this case an ABI PRISM 377 DNA Sequencer), the specific allele products can be differentiated from each other on the basis of which fluorescent dye they carry. This method can be used for both individual and pooled genotyping (Pastinen et al., 1996; Norton et al., 2002). Details of PCR primers and PCR reaction conditions and extension primers and extension reaction conditions for all SNPs are available on request.
D.W. Morris et al. / Schizophrenia Research 60 (2003) 167–172
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Table 1 SNPs from the DTNBP1 gene
A B C D E F G H
dbSNP IDa
Alternative marker nameb
Polymorphism
Frequencyc in controls
Frequencyc in casesd
p valuee
Frequencyc in casesf
p valueg
rs1047631 rs760666 rs2619539 rs3213207 rs2619542 rs2619550 rs2005976 rs760761
– P1287 P1655 P1635 – – P1757 P1320
A/G C/T G/C G/A C/T C/G A/G C/T
0.840 0.773 0.485 0.115 0.524 0.195 0.199 0.784
0.868 0.759 0.486 0.100 0.523 0.163 0.162 0.826
0.277 0.632 0.964 0.490 0.980 0.212 0.149 0.105
0.841 0.769 0.438 0.121 0.569 0.212 0.205 0.788
0.975 0.933 0.352 0.838 0.359 0.660 0.890 0.916
a
NCBI SNP Cluster ID, from the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/). Marker names used by Straub et al. for those SNPs genotyped in that study. c Of first allele shown. d All cases. e p value calculated for all cases and all controls. f Allele frequency for only those cases that are family history positive (n = 65; see Materials and methods). g p value calculated for cases that were family history positive and all controls. b
2.4. Statistical analysis
3. Results
SNPs were tested for association with the phenotype by using a 2 2 contingency table to calculate a v2 statistic. Haplotype analysis was performed using the HAPMAX programme (http://www.uwcm.ac.uk/ uwcm/mg/download). HAPMAX was first used to estimate marker – marker haplotype frequencies on transmitted and nontransmitted chromosomes in cystic fibrosis families (Krawczak et al., 1988). HAPMAX employs the EM algorithm to calculate haplotype frequencies. Intermarker linkage disequilibrium (LD) was measured using DV (Lewontin, 1964). DV values were calculated using the GOLD programme (http://www.sph.umich.edu/csg/abecasis/ GOLD/).
Of the 219 cases, 176 were diagnosed as schizophrenia and 43 as schizo-affective disorder. The frequencies of each SNP in the case and control groups are detailed in Table 1 along with the p value for each test of association. All SNPs were found to be in Hardy –Weinberg equilibrium in both the case and control samples. An exception to this was SNP A, which was not in Hardy –Weinberg equilibrium in either the cases or the controls ( p = 0.024 and p = 0.018, respectively). This is likely to be due to the low frequency of the minor allele in both samples. A comparison of the allele frequencies of the five SNPs (B, C, D, G and H in Table 1) genotyped in both studies shows that all five SNPs have strikingly
Fig. 1. Map of DTNBP1 showing the physical positioning of the eight SNPs (detailed in Table 1) genotyped in this study within the exonic structure of the gene. The intervals between adjacent SNPs are given in base pairs (bp).
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similar allele frequencies. In our study, the minor allele frequencies of SNPs B, C, D, G and H in the combined sample of cases and controls are 0.234, 0.485, 0.108, 0.181 and 0.196, respectively (separate frequencies for cases and controls are in Table 1). These compare to frequencies of 0.246, 0.463, 0.102, 0.167 and 0.178 in the total Straub sample (calculated using TRANSMIT; Straub et al., 2002b). None of the SNPs was found to be associated with illness. Of the five SNPs that were genotyped by Straub et al. in their sample, three of them (D, G and H) were found to be associated with schizophrenia when the sample was limited to one affected offspring per family under the ‘‘narrow’’ diagnostic category (Straub et al., 2002b). Haplotype analyses were performed with haplotypes constructed from adjacent markers in a sliding window fashion across the gene. HAPMAX was used to calculate haplotype frequencies for two-, three- and four-marker haplotypes. Association analyses were performed using the two-, three- and four-marker haplotypes but no evidence of association was found (data not shown). DVwas calculated for all pairs of SNPs in both the cases and the controls. These data are presented in Tables 2 and 3. The pattern of LD is broadly similar in both samples. SNPs B and D are both in very tight LD with all other SNPs across the gene. SNP A, the most telomeric SNP, is in moderate LD with the remaining SNPs. After that, there is a clear divide in the pattern of LD. There is strong LD between SNPs C and E, and between SNPs F, G and H, but there is only weak LD between these two sets of SNPs. Straub et al. (2002b) also reported pairwise DVvalues for all SNPs genotyped in their study. For SNPs that have been genotyped in both studies, DVvalues are very similar. For example, in our control sample, the DV values Table 2 DVvalues calculated for all pairs of SNPs (A – H) in the case sample
A B C D E F G H
A :::
B
1.00 0.62 0.92 0.54 0.62 0.62 0.63
::: 0.97 1.00 1.00 1.00 1.00 1.00
C
D
E
F
G
H
::: 1.00 1.00 0.26 0.23 0.23
::: 0.93 0.88 0.88 1.00
::: 0.24 0.21 0.21
::: 0.98 0.98
::: 1.00
:::
Table 3 DVvalues calculated for all pairs of SNPs (A – H) in the control sample
A B C D E F G H
A :::
B
0.99 0.35 0.86 0.30 0.50 0.50 0.49
::: 0.97 0.99 1.00 1.00 1.00 1.00
C
D
E
F
G
H
::: 1.00 1.00 0.02 0.03 0.02
::: 1.00 0.90 0.90 1.00
::: 0.03 0.01 0.03
::: 1.00 1.00
::: 1.00
:::
calculated for the SNP pairs B and C, C and G, and D and H are 0.97, 0.23 and 1.00, respectively. These correspond to values of 1.00, 0.15 and 0.93 in Straub et al. (2002b).
3. Discussion There are striking similarities in both the allele frequencies and DV values calculated for the SNPs genotyped both in this study and in that of Straub et al. (2002b). These similarities suggest that the samples used in both studies have been drawn from the same homogeneous population. Therefore, it is surprising that we do not identify the association reported by Straub et al. (2002b). There are a number of possible reasons for this. The first relates to known differences between the samples. Our sample was composed of individuals with schizophrenia drawn from the Irish population, whereas in the study of Straub et al. (2002b), cases were derived from pedigrees with multiply affected individuals. The genetic architecture of schizophrenia in such families may differ from that of unselected cases. Multiply affected families may represent a distinct genetic subset of schizophrenia. Earlier studies of schizophrenia based on a sporadic/familial distinction have been criticized as oversimplistic (Kendler, 1988). However, the assumption that most cases of schizophrenia share similar genetic aetiology may be equally simplistic. Such considerations may be relevant to the finding of association in Straub’s family-based sample and the absence of association in the present population-based sample. To examine this, we stratified our sample of cases into those that had a first or second degree
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relative with schizophrenia or schizo-affective disorder and those that did not. Association analysis was then performed in the SNP data using only those cases that were ‘family history positive’. Single marker analysis (see Table 1) and haplotype analysis (data not presented) provided no evidence of association and hence does not support the sporadic/familial divide. However, given the small sample size of the family history positive cases (n = 65), we cannot make any firm conclusions on this. A second possibility is that our study lacks power. Because the study of Straub et al. (2002b) does not include odds ratios/relative risk information for the associated SNPs D, G and H, we are unable to directly calculate the power of our sample to replicate their findings. However, we can address this issue indirectly by calculating odds ratios (OR) with 95% confidence intervals (95% CI) for SNPs D, G and H in our sample. These data are presented for both our full case-control sample and for the subset of family history positive cases in Table 4. Examining these data allows us to make certain post hoc observations. Firstly, from the full schizophrenia sample, we can exclude, with 95% certainty, an OR>1.34 for association with SNPs D, G or H. Secondly, examining the family history positive cases indicates that we can exclude, with 95% certainty, an OR>2 for the same SNPs. A third possibility relates to the differences in analytical methods employed. The findings of the Straub study are based on transmission distortion in data from nuclear families. The TDT test identifies linkage in the presence of association (Spielman et al., 1993), a finding that assumes the independence of the nuclear families. If this assumption is invalid and an unrecognised pedigree structure exists in a subset of the Straub et al. (1995) nuclear families, the reported
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association finding may reflect the previously reported linkage. This is probably unlikely given the large numbers of families recruited from throughout Ireland but would explain our failure to replicate the association. Alternatively, given that the findings of Straub et al. (2002b) are family-based, they may represent segregation distortion in this chromosomal region and hence evidence of association would not be found in our case-control sample. Finally, is it possible that we have missed the association? We have not genotyped every SNP from the Straub study in our sample. However, we have tested the three SNPs that provided the strongest evidence of association in that study. In addition, we have genotyped five other SNPs from across the gene. Single marker analysis of these SNPs does not suggest an association between variants in the DTNBP1 gene and schizophrenia in our sample. We performed haplotype analysis with haplotypes constructed from adjacent SNPs. These haplotypes span the region of strongest association as adjudged by haplotype analysis in the Straub study. Again, we found no evidence of association in our sample; hence, this explanation is possible but unlikely. Further studies on this and other samples will be required to determine which of the above explanations is most likely to establish the validity of the putative association between DTNBP1 and schizophrenia. In particular, we are unable to exclude the possibility that the DTNBP1 association is specific to a familial subtype of schizophrenia identified in these multiply affected pedigrees. This possibility must be tested in additional familial and population-based samples. Clarification of this issue could profoundly effect future study design and our understanding of the aetiology of schizophrenia.
Acknowledgements Table 4 Odds ratios with 95% confidence intervals from association study data SNP
D G H
Schizophrenia cases/controls
Family history positive cases/controls
OR
95% CI
OR
95% CI
0.86 0.78 0.76
0.55 – 1.34 0.54 – 1.11 0.54 – 1.07
1.06 1.03 0.97
0.56 – 2.00 0.62 – 1.71 0.59 – 1.60
The authors gratefully acknowledge the contribution of all participating individuals and institutions in this study. Dr. Morris is supported by the Higher Education Authority. Dr. Corvin is a Wellcome Trust Research Fellow in Mental Health. The authors would like to thank the Health Research Board (Ireland) and the Stanley Medical Research Institute (USA) for their support of this research.
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