Association study of CHRFAM7A copy number and 2bp deletion polymorphisms with schizophrenia and bipolar affective disorder.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:571 –575 (2006) Association Study of CHRFAM7A Copy Number and 2bp Deletion Polymorphisms With Schizophrenia and Bipolar Affective Disorder Rachel H. Flomen,1* David A. Collier,1,2 Sarah Osborne,1 Janet Munro,1 Gerome Breen,1,2 David St Clair,3 and Andrew J. Makoff1 1 Division of Psychological Medicine, Institute of Psychiatry, King’s College London, London, United Kingdom MRC Social Genetic and Developmental Psychiatry Research Centre, Institute of Psychiatry, King’s College London, London, United Kingdom 3 Department of Mental Health, Institute of Medical Science, University of Aberdeen, Aberdeen, Scotland, United Kingdom 2 Schizophrenia and bipolar disorder are major psychiatric diseases that have a strong genetic element. Markers in the vicinity of the CHRNA7 gene at 15q13-q14 have been linked with an endophenotype of schizophrenia, P50 sensory gating disorder, with schizophrenia itself and with bipolar disorder. We have measured the copy number of the polymorphic partial duplication of CHRNA7 (CHRFAM7A) and genotyped a polymorphic 2bp deletion within exon 6 of CHRFAM7A. In this study, 208 probands with a primary diagnosis of schizophrenia, 217 with a diagnosis of bipolar affective disorder and 28 with schizoaffective or other psychotic disorders were examined together with 197 controls recruited from the same region in Scotland. No significant association was seen for schizophrenia and bipolar disorder by genotype or allele overall for either polymorphism, but a mildly significant association by genotype (P ¼ 0.04) was observed for absence of CHRFAM7A when the sample was analyzed as a single psychosis phenotype. ß 2006 Wiley-Liss, Inc. KEY WORDS: copy number polymorphism; CHRNA7; CHRFAM7A; schizophrenia; bipolar disorder Please cite this article as follows: Flomen RH, Collier DA, Osborne S, Munro J, Breen G, St Clair D, Makoff AJ. 2006. Association Study of CHRFAM7A Copy Number and 2bp Deletion Polymorphisms With Schizophrenia and Bipolar Affective Disorder. Am J Med Genet Part B 141B:571–575. INTRODUCTION The alpha-7 nicotinic cholinergic receptor subunit (CHRNA7) gene is widely expressed in the central nervous system, with high levels in the hippocampus. It maps to chromosome 15q13-q14 [Chini et al., 1994], a region that has *Correspondence to: Rachel H. Flomen, Division of Psychological Medicine, Institute of Psychiatry, King’s College London, 1 Windsor Walk, Denmark Hill, London SE5 8AF, UK. E-mail: email@example.com Received 28 July 2005; Accepted 17 February 2006 DOI 10.1002/ajmg.b.30306 ß 2006 Wiley-Liss, Inc. been implicated in several neuropsychiatric disorders, including epilepsy and psychosis, by linkage and association study. Markers at or near CHRNA7 show evidence of strong linkage to a secondary phenotype of schizophrenia, P50 [Freedman et al., 1997; Leonard et al., 1998], which compares the amplitudes of the responses after 50 msec to two auditory signals 500 msec apart. In most individuals the second response is attenuated, which is believed to be a measure of the ability to filter out repetitive stimuli. In schizophrenic patients and around half of their first-degree relatives, the normal attenuation of P50 response is reduced and this effect is normalized by nicotine and atypical antipsychotics such as clozapine [Freedman et al., 1997]. Linkage to P50 mapped at 15q13-q14 with a peak LOD score of 5.3 at a marker within intron 2 of CHRNA7, so that P50 appears to segregate as a Mendelian trait with strong evidence of linkage to this gene [Freedman et al., 1997]. Not surprisingly for a complex disorder, it has proved more difficult to demonstrate strong linkage of this region to schizophrenia itself, with the same study having a much lower LOD score of 1.33 [Freedman et al., 1997]. Many other studies have reported weak evidence for linkage to schizophrenia [Kaufmann et al., 1998; Leonard et al., 1998; Riley et al., 2000; Gejman et al., 2001; De Luca et al., 2004] and others had negative findings [Neves-Pereira et al., 1998; Curtis et al., 1999; Meyer et al., 2002]. Significant linkage has been demonstrated with bipolar affective disorder [Edenberg et al., 1997; Turecki et al., 2001], where abnormal P50 has also been described [Baker et al., 1990]. These linkage studies have been supported by the observation that schizophrenia and bipolar disorder are associated with 15q13-q14 markers [Stassen et al., 2000]. The region has also been linked to two idiopathic epilepsies [Elmslie et al., 1997; Neubauer et al., 1998]. The 15q13-q14 region is highly duplicated and this has hindered mapping, especially with earlier incorrectly assembled sequence data for this region. More recent assemblies (e.g., NT_010194 which had this region correctly assembled since version 15, build 33 available in April 2003) now confirm and extend the map produced in this laboratory [Riley et al., 2002]. Exons 5–10 of CHRNA7 are located at one end of a 300 kb duplicon, with the promoter and exons 1–4 in a unique region. In the other 300 kb duplicon, exons 5–10 are distal to exons D-A which are duplicated from an unknown gene FAM7A in different duplicons. This forms a hybrid gene (CHRFAM7A), which is transcribed, although translation is uncertain [Gault et al., 1998; Riley et al., 2002]. These duplicons are polymorphic, due to copy number polymorphisms (CNPs). Chromosomes with one or no copies of the hybrid CHRFAM7A have so far been identified [Riley et al., 2002; Taske et al., 2002], as have two or three copies of FAM7A in the few individuals represented in the sequence databases. CHRFAM7A additionally carries a polymorphic 2bp deletion 572 Flomen et al. within exon 6, which, if translated, is predicted to result in a truncated protein [Gault et al., 1998]. The presence of CHRFAM7A in many but not all human genomes as well as the near 100% identity of the 30 part in common with CHRNA7 strongly suggests that its acquisition is a recent event in human evolution [Riley et al., 2002; Taske et al., 2002]. This view is supported by the failure to detect CHRFAM7A in chimpanzee DNA by FISH mapping [Locke et al., 2003] and in the chimpanzee sequence database [http:// www.ncbi.nlm.nih.gov/genome/guide/chimp/]. The high frequencies of CHRFAM7A in a variety of populations suggest that it, or a subsequent event such as the 2bp deletion, may confer a selective advantage. In order to clarify the role of CHRFAM7A and its 2bp deletion in susceptibility to psychosis, we have investigated these two polymorphisms for association with schizophrenia and bipolar disorder in a large sample of white Caucasians. We have used robust assays to investigate the 2bp deletion in combination with CHRFAM7A copy number and these identify three CHRFAM7A alleles (null, wild-type, and 2bp deletion) in a total of six possible genotypes. MATERIALS AND METHODS Clinical Samples Four hundred and fifty three probands with a major psychosis were analyzed together with 197 ethnically matched controls collected from the same geographical region of Scotland [McGinnis et al., 2001; Stefansson et al., 2003; NevesPereira et al., 2005]. Subjects were recruited through Scottish psychiatric hospitals and met DSM111R criteria for schizophrenia or schizoaffective disorder and DSM1V for bipolar 1 disorder. Consensus diagnosis was made by two consultant psychiatrists based on a combination of examination of psychiatric case notes and clinical interview. The psychosis sample was clinically classified as follows: 208 schizophrenia, 217 bipolar disorder, and 28 other psychoses (including 11 with schizoaffective disorder). Controls were recruited via the Scottish blood transfusion service from the same region of Scotland and were 57% male and 43% female. As the incidence of psychosis in the general population is low, they were not specifically screened for mental illness. In the UK only healthy people not taking medication can donate blood, and therefore these controls are unlikely, if at all, to include a significant number of psychotic individuals. All individuals gave written consent to participate and Multiregional Ethics Committee (MREC) approval was granted for this study. Genotyping Genotyping for the 2bp deletion polymorphism was by limited cycle fluorescent PCR (21 cycles of 948C/30 sec, 588C/ 30 sec, 728C/1.5 min) using primers flanking the 2bp deletion. 2bpForFAM GGGCATATTCAAGAGTTCCTGCTAC and 2bpRev CCACTAGGTCCCATTCTCCATTG gave a product size of 170 bp in the absence of the deletion and 168 bp in its presence. PCR products were resolved using a 3100 fluorescent genotyper and Genemapper v3.0 software to ascertain 170 bp:168 bp ratios. Each assay was performed at least twice. Copy number of CHRFAM7A was determined with a Taqman assay spanning the breakpoint between FAM7A exons DA and CHRNA7 exons 5–10 (forward primer: AGTAATAGTGTAATACTGTAACTTTAAAATGTGTTACTTGT, reverse primer: AGCCGGGATGGTCTCGAT, MGB labeled probe: TCCT GACTGTACACATAAAA). Ct values were determined on three separate occasions for the CHRFAM7A specific assay and compared with Ct values for an endogenous control, the beta-2- microglobulin gene (forward primer: TGGGTTTCATCCATCCGACATT, reverse primer: AGACAAGTCTGAATGCTCCAC TTTT, MGB labeled probe: ATTCTTCAGTAAGTCAACTTC). The differential value between test and control was used to determine copy number of the CHRFAM7A duplicon. Taqman results were interpreted using values corresponding to 0, 1, or 2 copies of CHRFAM7A which was determined empirically using 86 samples of known copy number in addition to 11 members of 2 families which included homozygous null and obligate heterozygous individuals [Riley et al., 2002; Taske et al., 2002]. RESULTS The presence or absence of CHRFAM7A, containing exon 6 either identical to that in CHRNA7 or with the 2bp deletion, results in three alleles and six possible genotypes (Table I). All samples were initially assayed for the 2bp deletion by measuring the ratio of exon 6 copy number with and without the deletion. For three of the six genotypes, the assay identifies CHRFAM7A copy number. These three genotypes had two product sizes with informative ratios: 1:1 ¼ 2 copies of CHRFAM7A, both with the 2bp deletion; 3:1 ¼ 2 copies of CHRFAM7A, 1 with the 2bp deletion; 2:1 ¼ 1 copy of CHRFAM7A with the 2bp deletion (see Table I). Samples lacking the 2bp deletion on both chromosomes had only one product size and were therefore uninformative for CHRFAM7A copy number, which was determined using the Taqman assay. Results from the Taqman and 2bp fluorescent assays were then combined to identify all six genotypes. This combined assay was validated for CHRFAM7A copy number in preliminary experiments which showed that the three genotypes informative in the 2bp deletion assay generated the expected CHRFAM7A copy numbers using Taqman assay in 90 out of 91 comparisons. This single discrepancy was due to Taqman values predicting CHRFAM7A copy number in between 1 and 2. The genotype frequencies for schizophrenia, bipolar, and controls are shown in Table II, with patient numbers given from both assays separately (a and b) and combined (c). Comparison of genotype frequencies between schizophrenia or bipolar samples and controls show no significant difference, either analyzed separately (w2 ¼ 8.00, 5 df, P ¼ 0.2; w2 ¼ 6.96, 5 df, P ¼ 0.2), or in the combined psychosis phenotype (w2 ¼ 7.26, 5 df, P ¼ 0.2). The genotype data were also analyzed to investigate whether (1) CHRFAM7A copy number, (2) presence of wild-type CHRFAM7A, or (3) presence of the 2bp deletion were associated with any of the psychosis phenotypes. When copy number of CHRFAM7A was considered alone, there were small differences between schizophrenia or bipolar probands and controls (w2 ¼ 3.85, 2 df, P ¼ 0.2; w2 ¼ 4.93, 2 df, P ¼ 0.09), but when all psychosis patients were combined there was a small significant difference from controls (w2 ¼ 6.26, 2 df, TABLE I. Six Possible Genotypes for CHRFAM7A þ 2bp Deletion CHRNA7a Genotype 11 12 22 13 23 33 CHRFAM7Aa Wt Wt 2 2 2 2 2 2 0 1 2 0 1 0 2bp deletion Wt:2bp deletion 0 0 0 1 1 2 1 peak 1 peak 1 peak 2:1 3:1 1:1 Allele 1 ¼ absence of CHRFAM7A, allele 2 ¼ presence of wild-type (wt) CHRFAM7A, allele 3 ¼ presence of CHRFAM7A with 2bp deletion in exon 6. a Number of copies of exon 6. Association of CHRFAM7A Polymorphism and Psychosis 573 TABLE II. Genotype Frequencies for Schizophrenia, Bipolar, and Control Samples. (a) Patient counts from 2bp assay, (b) patient counts from taqman assay, (c) combined patient counts. Alleles are defined as in Table I (a) 2bp assay (all samples) No. of peaks 1 2 2 2 2 (2:1) (3:1) (1:1) (unclear) Genotype Genotype 11 12 22 13 23 33 Total Schizophrenia Bipolar Others 11,12, or 22 76 76 82 13 11 8 23 23 73 72 83 33 32 27 25 13, 23, or 33 3 10 3 Total 195 193 216 (b) Taqman assay (only samples with 1 peak on 2bp assay) CHRFAM7A copy no. 0 1 2 Unclear Controls Genotype 11 12 22 11, 12, or 22 Total Controls Schizophrenia 3 19 49 5 76 (c) Combined results 11 2 7 5 1 26 Bipolar Others 1 33 34 8 76 1 28 53 0 82 0 4 6 1 11 Controls Schizophrenia Bipolar Others Psychosis 3 (0.02) 19 (0.10) 49 (0.26) 11 (0.06) 73 (0.39) 32 (0.17) 187 1 (0.01) 33 (0.19) 34 (0.19) 8 (0.05) 72 (0.41) 27 (0.15) 175 1 (0.00) 28 (0.13) 53 (0.25) 23 (0.11) 83 (0.39) 25 (0.12) 213 0 (0.00) 4 (0.17) 6 (0.25) 2 (0.08) 7 (0.29) 5 (0.21) 24 2 (0.00) 65 (0.16) 93 (0.23) 33 (0.08) 162 (0.39) 57 (0.14) 412 Overall genotype comparisons: schizophrenia versus control: w2 ¼ 8.00, 5 df, P ¼ 0.2, bipolar versus controls: w2 ¼ 6.96, 5 df, P ¼ 0.2, psychosis versus controls: w2 ¼ 7.26, 5 df, P ¼ 0.2. CHRFAM7A copy number comparisons (11 vs. 12/13 vs. 22/23/33): schizophrenia versus controls: w2 ¼ 3.85, 2 df, P ¼ 0.06, bipolar versus controls: w2 ¼ 4.93, 2 df, P ¼ 0.09, psychosis versus controls: x2 ¼ 6.26, 2 df, P ¼ 0.04. CHRFAM7A wt comparisons (22 vs. 12/23 vs. 11/13/33): schizophrenia versus controls: w2 ¼ 4.40, 2 df, P ¼ 0.1, bipolar versus controls: w2 ¼ 0.34, 2 df, P ¼ 0.8, psychosis versus controls: w2 ¼ 1.84, 2 df, P ¼ 0.4. 2bp deletion comparisons (33 vs. 13/23 vs. 11/12/22): schizophrenia versus controls: w2 ¼ 0.19, 2 df, P ¼ 0.9, bipolar versus controls: w2 ¼ 2.52, 2 df, P ¼ 0.3, psychosis versus controls: w2 ¼ 1.12, 2 df, P ¼ 0.6. P ¼ 0.04). However, this mild significance is lost after correction for multiple testing. We found no evidence for association of wild-type CHRFAM7A with schizophrenia (w2 ¼ 4.40, 2 df, P ¼ 0.1), bipolar disorder (w2 ¼ 0.34, 2 df, P ¼ 0.8) or combined psychosis (w2 ¼ 1.84, 2 df, P ¼ 0.4). Similarly we found no evidence for association of the 2bp deletion alone with any of the three phenotypes (w2 ¼ 0.19, 2 df, P ¼ 0.9; w2 ¼ 2.52, 2 df, P ¼ 0.3; w2 ¼ 1.12, 2 df, P ¼ 0.6). The overall genotype frequencies for controls and bipolar probands were in Hardy–Weinberg equilibrium (w2 ¼ 2.02, 3 df, P ¼ 0.6; w2 ¼ 3.00, 3 df, P ¼ 0.4), while schizophrenia probands deviated from expected values (w2 ¼ 14.23, 3 df, P ¼ 0.003). However, when the copy number of CHRFAM7A was considered alone all were in Hardy–Weinberg equilibrium (controls: w2 ¼ 1.33, 1 df, P ¼ 0.3; bipolar: w2 ¼ 2.09, 1 df, P ¼ 0.2; schizophrenia: w2 ¼ 1.14, 1 df, P ¼ 0.3), while combined psychosis was of borderline significance (w2 ¼ 3.84, 1 df, P ¼ 0.05). Failures or ambiguities in either assay are potential confounders of the validity of the combined assay as they differentially affect the relative proportions of two groups of genotypes. Where a sample was omitted due to the Taqman assay failing to estimate a copy number, this results in the affected genotypes (11, 12, and 22) being under-represented. Conversely, where the 2bp deletion assay produced two peaks that failed to give a clear value for their ratios (e.g., between 2:1 and 3:1), omission of these samples results in the other group of genotypes (13, 23, and 33) being under-represented. To check whether these omitted samples significantly affected the outcome, genotype counts were adjusted to maintain the ratio between the two groups of genotypes (13 þ 23 þ 33/ 11 þ 12 þ 22) as indicated by the 2bp deletion assay (data not shown). For all three patient groups, the adjustment was very small, with no more than one patient count added to or subtracted from each genotype group. This had only a small effect on all of the comparisons, with the association of combined psychosis with CHRFAM7A copy number of borderline significance (w2 ¼ 5.96, 2 df, P ¼ 0.05). DISCUSSION The 2bp deletion polymorphism is in exon 6 of CHRFAM7A, which is itself polymorphic, existing as a null allele or single copy allele. The 2bp deletion can clearly only occur with the single copy allele of the CHRFAM7A CNP, and may be present in one (genotype 23) or both (genotype 33) of two copies of CHRFAM7A, or in the only copy (genotype 13) of CHRFAM7A (see Table I). It is therefore important that both polymorphisms are considered together. We have achieved this by combining a fluorescent PCR assay with a robust Taqman assay to determine copy number accurately. Our data show no evidence for schizophrenia or bipolar disorder separately or as a combined psychosis phenotype being associated with the two CHRFAM7A polymorphisms together. This agrees with a French study using a much smaller sample size (70 cases and 77 controls) which also found no association with schizophrenia [Raux et al., 2002]. A Chinese group investigated the 2bp deletion polymorphism without considering the CHRFAM7A CNP. They reported weak association for presence of the 2bp deletion with bipolar 574 Flomen et al. disorder (P ¼ 0.044) using 77 cases and 135 controls [Hong et al., 2004], but no association with schizophrenia using 146 cases and 151 controls [Lai et al., 2001]. The French group also found no association of the 2bp deletion with schizophrenia, although they did find an association with abnormal P50, which was mostly limited to the control group [Raux et al., 2002]. We found no evidence for association between 2bp deletion and any psychosis phenotype, but have not investigated the P50 endophenotype in our Scottish cohort. We will be performing association studies with P50 on a sample of patients and their families attending the Maudsley Hospital, when the electrophysiological data is complete. The Chinese group also found evidence for more than two alleles of the 2bp deletion genotype in 3 out of 77 bipolar cases [Hong et al., 2004], implying either a copy number of more than one per chromosome for the CHRFAM7A CNP or the presence of the 2bp deletion on CHRNA7. We have examined over 600 patient and control samples here and an additional 480 samples in other studies (R. Flomen, unpublished observations) but have never observed a ratio of wt/2bp<1. As all our DNA samples are from white Caucasians, this difference may be due to ethnicity, although methodological differences cannot be excluded. Our data produced weak evidence for a combined psychosis phenotype being associated with only one copy of CHRFAM7A (P ¼ 0.04), regardless of presence or absence of the 2bp deletion. This observation clearly needs to be treated with considerable caution, as the association is lost following Bonferroni correction for multiple testing. Whether there is a stronger association with no copies of CHRFAM7A could not be determined, as this genotype is too rare to draw any conclusion. Other studies have similarly found that this genotype is very rare [Gault et al., 1998; Raux et al., 2002; Li et al., 2004]. As discussed above, we found no evidence for association with the 2bp deletion, and we also failed to find association of any psychosis phenotype with the presence of a wild-type CHRFAM7A. If translated in the same cell, the CHRFAM7A translation products might interact with those of CHRNA7 and disrupt formation of the alpha-7 receptor homopentamer, acting in a dominant negative manner [Raux et al., 2002]. This could be prevented by the 2bp deletion, which would cause a truncated product to be formed. Consequently either polymorphism could contribute to pathogenesis of the major psychoses linked to 15q13-q14. Our suggestive evidence for reduced levels of CHRFAM7A in psychosis is inconsistent with deleterious interaction with CHRNA7 polypeptides being the diseaseassociated mechanism. Indeed, our findings cannot be reconciled with any role for the CHRFAM7A gene product in psychosis, although it may be involved in other phenotypes such as cognitive functions in psychosis patients or even in the general population. If CHRFAM7A protein were implicated in schizophrenia or bipolar disorder, absence of the gene product would affect the phenotype irrespective of whether this was a consequence of the CHRFAM7A null allele or of the 2bp deletion. However, only the absence of one copy of the CHRFAM7A gene appears to be a risk allele. Therefore if the weak association of CHRFAM7A copy number with psychosis is real, our data suggest that the mechanism involves transcription rather than translation of CHRFAM7A. One possible mechanism might be competition between the two genes for a transcription factor that binds to a sequence element in the shared part of each gene. However, examination of this region for repetitive elements on the UCSC database (www.genome.ucsc.edu) has failed to identify a plausible candidate (data not shown). Another possibility might be production of anti-sense RNA by transcription of the anti-sense strand of CHRFAM7A, thereby modulating expres- sion of CHRNA7. However, this is unlikely as all published CHRFAM7A cDNAs are transcribed on the sense strand [Riley et al., 2002]. As CHRFAM7A is located on a 300 kb duplicon, the CHRFAM7A CNP may affect a much larger region than the gene itself. We have evidence that it covers a region in the range of 380–560 kb in 15q13-q14 (in preparation). Therefore, any gene in this region would also have its copy number altered by this CNP and potentially may have a role in the phenotype. Six genes of unknown function have been identified in this region with the following accession numbers: CR749235, AL117445, AK131275, AK090401, AK090403 (www.genome.ucsc.edu). Finally, it is possible that the CHRFAM7A CNP is in linkage disequilibrium with the functional polymorphism responsible for predisposition to psychosis. Abundance of CHRFAM7A in several human populations and its absence in other higher primates suggests that it may have an evolutionarily beneficial effect. Our data demonstrating a weak association of reduced levels of CHRFAM7A with psychosis are consistent with this view. Many more studies on this complex region on chromosome 15 are necessary to resolve these issues. Accordingly, we are currently investigating CHRFAM7A and other variants on 15q13-q14 for possible association with psychosis and with endophenotypes such as P50. ACKNOWLEDGMENTS This work was supported by a NARSAD Independent Investigator Award. 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