LS Carroll, KM Kendall, MC O'Donovan, MJ Owen and NM Williams Evidence that putative ADHD low risk alleles at SNAP25 may increase the risk of schizophrenia LS Carroll, KM Kendall, MC O'Donovan, MJ Owen and NM Williams Department of Psychological Medicine and Neurology, Cardiff University, UK BACKGROUND Schizophrenia is a common, complex disorder affecting ~1% of the population. The estimated heritability is >80%, a significant proportion of which is likely due to polygenic factors.1 One of the best supported candidate schizophrenia susceptibility genes is dysbindin (DTNBP1).2 Altered expression of DTNBP1 has been shown to cause variation in levels of synaptosomal associated protein 25 kDa (SNAP25).3 SNAP25 is an attractive candidate schizophrenia susceptibility gene given that multiple studies have found altered SNAP25 transcript or protein levels in schizophrenia.4,5 The gene may also be involved in the mechanism of action of antipsychotic medication.6 SNAP25 is an important member of the SNARE complex, a structure that mediates synaptic vesicle exocytosis.7 In order to test the hypothesis that SNAP25 is a schizophrenia susceptibility gene, we carried out mutation screening of SNAP25 exons and the immediate 5’ flanking region and examined the observed SNPs in a UK case control sample. We followed this by genotyping an additional 31 informative tag SNPs spanning the SNAP25 locus in the same sample. METHODS SNAP25 (20p12.2) has two major protein isoforms, differing by alternate 118 bp coding exons at exon 5. We performed mutation screening of SNAP25 in 14 unrelated individuals with schizophrenia (7 males, 7 females) selected at random from our UK association sample. This gives 80% power to detect alleles with a frequency of 0.05 and 95% power to detect alleles with a frequency of 0.10. Amplimeres were screened by high resolution DNA melting analysis (Light-Scanner system) and suspected DNA variants characterised by BigDye DNA sequencing. We examined the seven SNPs emerging from the mutation screen, then a further 31 informative tag SNPs in a UK case control sample. This consisted of 662 cases (448 males, 214 females) with a diagnosis of schizophrenia according to DSM-IV criteria and 716 controls (482 males, 234 females), all of whom were unrelated and of white European descent. Genotyping was performed using the Sequenom MassARRAY system and Sequenom iPlex GOLD chemistry. Quality control involved analysis in the CEU HapMap Project individuals prior to genotyping in the case control sample.8 RESULTS We screened 3,965 bases at the SNAP25 locus in 14 unrelated individuals with schizophrenia from the UK. We failed to screen the final 229 bases of the universal 3’ UTR. We identified seven SNPs, six of which were listed in dbSNP build 128. A novel SNP (dbSNP ID ss107056528) was discovered in the putative promoter region (G/T transversion). Genotyping these SNPs in our case control sample revealed significant evidence for allelic association at rs3746544 (P = 0.004, OR = 1.26) and rs8636 (P = 0.003, OR = 1.27). Both remained significant (P = 0.02) after correction for multiple testing but were almost perfectly correlated (D' = 1, r2 = 0.997). We looked for further evidence of association by using a more comprehensive set of tag SNPs. We established the genotypes of the seven SNPs identified during mutation screening in the HapMap CEU samples. We merged these with those of the HapMap SNPs that span the SNAP25 locus. We used Tagger to select 31 tag SNPs. When combined with the six original independent SNPs, these captured 79% of the 131 SNPs genotyped in the HapMap CEU samples at the locus (r2 > 0.8, MAF >0.01). Genotyping these 31 extra SNPs in the case control sample revealed a nominally significant association at an additional five SNPs. The strongest evidence for association was at rs3787283 (P = 0.006, OR = 1.25). However, none survived correction for multiple testing. Figure 1. The 38 SNPs genotyped with those identified by mutation screening highlighted. Inter-marker LD is based on D’. The relative location of the two SNAP25 isoforms are shown at the top of the figure (NCBI Human Genome Build 36). DISCUSSION We investigated whether genetic variation at the SNAP25 locus might be associated with schizophrenia. Two of the seven SNPs identified during mutation screening exhibited allelic association with schizophrenia at a level that survived correction for the primary analysis. Genotyping an additional 31 tag SNPs revealed an extra five SNPs nominally associated with schizophrenia. However, none were stronger than that observed in the primary analysis. Our results should be considered as hypothesis generating and require follow up. We compared our results to studies reporting evidence for association with (i) antipsychotic response in schizophrenia and (ii) ADHD. Carriers of the allele which showed the strongest association in our study (G allele at rs3746544) show significantly poorer clinical response to antipsychotic treatment than non-carriers.6 Genetic variation at SNAP25 may confer an increased risk of developing a more severe form of schizophrenia which is more refractory to treatment. Alternatively, our association may reflect an impact of SNAP25 on treatment response. Two of the SNPs nominally associated with schizophrenia in our study (rs3787283, rs3746544) have been found to be associated with ADHD but in the reverse allelic direction i.e. risk alleles for schizophrenia are protective in ADHD.9,10 These findings are intriguing. Treatment of ADHD is based upon accentuating dopamine transmission with stimulants while the mainstay of treatment for schizophrenia is attenuating it with dopamine blockers. ACKNOWLEDGEMENTS: This work was funded by a Wellcome Trust Project Grant. KM Kendall was funded by a Wellcome Trust Vacation Scholarship. FURTHER INFORMATION: kimberleymkendall@googlemail.com http://medicine.cf.ac.uk/en/departments/psych-med-neurol/ REFERENCES 1. International Schizophrenia Consortium. 2009. Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature 460, pp 748-752 2. Williams NM, O'Donovan MC and Owen MJ. 2005. Is the dysbindin gene (DTNBP1) a susceptibility gene for schizophrenia. Schizophr Bull 31(4), pp 800-805 3. Numakawa T, Yagasaki Y, Ishimoto T et al. 2004. Evidence of novel neuronal functions of dysbindin, a susceptibility gene for schizophrenia. Hum Mol Genet 13(21), pp 2699-2708 4. Thompson PM, Kelley M, Yao J et al. 2003. Elevated cerebrospinal fluid SNAP-25 in schizophrenia. Biol Psychiatry 53(12), pp 1132-1137 5. Fatemi SH, Earle JA, Stary JM et al. 2001. Altered levels of the synaptosomal associated protein SNAP-25 in hippocampus of subjects with mood disorders and schizophrenia. 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