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The synaptosomal associated protein of 25 kDa (SNAP-25) gene, located on chromosome 20 p12-12p11.2 encodes a presynaptic terminal protein. SNAP-25 is differentially expressed in the brain, and primarily present in the neocortex, hippocampus, anterior thalamic nuclei, substantia nigra and cerebellar granular cells. Recently, a family-based genetic association was reported between variation in intelligence quotient (IQ) phenotypes and two intronic variants on the SNAP-25 gene. The present study is a follow-up association study in two Dutch cohorts of 371 children (mean age 12.4 years) and 391 adults (mean age 36.2 years). It examines the complete genomic region of the SNAP-25 gene to narrow down the location of causative genetic variant underlying the association. Two new variants in intron 1 (rs363043 and rs353016), close to the two previous reported variants (rs363039 and rs363050) showed association with variation in IQ phenotypes across both cohorts. All four single nucleotide polymorphisms were located in intron 1, within a region of about 13.8 kbp, and are known to affect transcription factor-binding sites. Contrary to what is expected in monogenic traits, subtle changes are postulated to influence the phenotypic outcome of complex (common) traits. As a result, functional polymorphisms in (non)coding regulatory sequences may affect spatial and temporal regulation of gene expression underlying normal cognitive variation.
Cognitive ability is currently considered as a polygenic trait influenced by many genes of moderate to small effect that in turn may interact with each other and with environmental factors (Butcher et al. 2006; Plomin & Spinath 2004; Savitz et al. 2006). Identifying the actual genes underlying normal cognitive variation has proven to be a daunting task, mainly because of this polygenic nature. So far, successful identification of genes underlying genetic variation in human cognitive ability has been mainly limited to mutations for relatively rare neurological disorders with considerably severe cognitive effects in which mental retardation or milder forms of cognitive disability are part of a syndromic phenotype [i.e. fragile X syndrome (Verkerk et al. 1991), Apert syndrome (Ibrahimi et al. 2005), Rett syndrome (Neul & Zoghbi 2004)]. These mutations occur generally in key regulatory proteins within general neuronal signaling pathways.
We recently conducted a family-based association study using an indirect (tagging) approach that involved the SNAP-25 gene and psychometric intelligence scores as a measure of cognitive ability in humans (Gosso et al. 2006a). Psychometric intelligence tests consist of a number of component subtests that taken together are used to infer a general intelligence quotient (IQ) score. Two single nucleotide polymorphisms (SNPs) in the SNAP-25 gene showed a highly significant association with IQ. Both were (non)coding variants. Associations in a (non)coding region of SNAP-25 can arise from variants in intronic and untranslated regions (UTR) that influence gene expression [e.g. variants located on promoter regions, transcription starting sites and 3′ UTR microRNA target sites], which in turn might result in individual variation among IQ phenotypes.
The initial analyses (Gosso et al. 2006a) were based on a tagging approach. We here perform follow-up analyses to (1) narrow down the location of causative genetic variant underlying the association in intron 1 and (2) identify extra regions on SNAP-25 gene not tagged during the previous analyses. Two independent extended cohorts of children (mean age 12.4 years) and adults (mean age 36.2 years) were used in order to identify these putative regulatory genomic variants underlying variation among IQ phenotypes.
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- Materials and methods
To continue our investigation of the possible role of the SNAP-25 gene in intelligence, we employed a family-based genetic association test in two independent cohorts of 371 children (mean age 12.42 years), and 391 adults (mean age 36.25 years). The selected SNPs gave a dense coverage of the first intron of SNAP-25, which was previously reported to be associated with intelligence (Gosso et al. 2006a). Single and haplotype analysis was conducted in the present study in order to (1) narrow down the location of causative genetic variant underlying the association in intron 1 and (2) identify extra regions on SNAP-25 gene not tagged during the previous analyses. Four SNPs (rs363039, rs363043, rs363016 and rs363050) located in intron 1, showed significant association with IQ phenotypes. Haplotype analysis confirmed the single association results. Combined data across age cohorts showed highly significant associations among IQ phenotypes for both G-T-T [PIQ χ2(1) = 8.27, P = 0.004] and G-C-T [VIQ χ2(1) = 8.61, P = 0.003] haplotypes. Interestingly, two haplotypes were independently found associated to IQ phenotypes among young and adult cohorts. Within the young cohort, G-T-T was the strongest associated haplotype [PIQ χ2(1) = 9.36, P = 0.002], whereas G-C-T showed the strongest association among the adult cohort [FSIQ χ2(1) = 10.08, P = 0.001]. Variance in these haplotypes accounts for 1% and 3% of the phenotypic variance in PIQ and FIQ, respectively.
Such differential genotypic effects might be possibly explained within a heterogeneous genomic context. Although physical TFBS are a constitutive portion of the genome, the cellular and genomic context will determine whether a given TF sequence(s) become functional or not. Transcription factors are differentially expressed in response to developmental requirements, and even more important, like QTL, single TF will not be sufficient to trigger a regulatory response, but they will rather interact in a collaborative manner. For example, low levels of TF p53 are constitutively expressed in the developing nervous system (embryonic and neonatal) under normal growth conditions and this is downregulated in adults (Komarova et al. 1997). Nevertheless, role of p53 in differentiation rather than apoptosis during sensory neuronal development still has to be determined.
Likely, IQ can be considered a truly polygenic complex trait, and as such, not a single common allelic variant might be involved in IQ phenotype variation, but rather, similar genetic effects might be exerted by diverse allelic variants. Alternatively, our results could indicate that the causal variant is older than the SNPs that have been tested and in fact is present on both haplotypes. It is worth noting that rare alleles may still contribute importantly to variation in cognitive ability, albeit their small effects can be only identified within a multicenter collaborative study framework, mainly because of the relative large amount of samples required to achieve power to detect their genetic effects.
The SNAP-25 gene, located on chromosome 20 p12-12p11.2 encodes a presynaptic terminal protein. SNAP-25 is thought to be differentially expressed in the brain and is primarily present in the neocortex, hippocampus, anterior thalamic nuclei, substantia nigra and cerebellar granular cells. In the mature brain, expression is mainly seen at presynaptic terminals (Oyler et al. 1989). Two splicing variant of the SNAP-25 exist, SNAP-25a and SNAP-25b isoforms (Bark & Wilson 1991). During development, SNAP-25a isoform is involved in synaptogenesis, forming presynaptic sites and neuritic outgrowth (Osen-Sand et al. 1993; Oyler et al. 1989), whereas in the mature brain, the SNAP-2b isoform forms a complex with syntaxin and the synaptic vesicle proteins (synaptobrevin and synaptotagmin) that mediates exocytosis of neurotransmitter from the synaptic vesicle into the synaptic cleft (see Bark & Wilson 1994; Horikawa et al. 1993; Low et al. 1999; Seagar & Takahashi 1998).
SNAP-25 isoforms (SNAP-25a and SNAP-25b) are fundamental for keeping a balanced trade-off between synaptic formation and neurotransmitter vesicle release; however, evolutionary (comparative genomics) analysis of the coding sequence showed no selection in favor of any of the gene-coding variants on SNAP-25. If variation of coding variants may not per se be associated to phenotypic variation, then, the next possible scenario might be the presence of regulatory effects exerted by variants on (non)coding regions. Regulatory (non)coding variants may interact in a concerted manner rather than in isolation, with the capacity to regulate gene expression. Genetic (non)coding variants present within intron 1 might be involved in regulation of protein isoforms expression. All associated SNPs were involved in TFBS changes (gain/loss of TFBS). Furthermore, because functional TFBS are predicted to interact in a co-operative manner rather than in isolation, a global overview might be required to (1) identify known and unknown TFBS and (2) putative functional (non)coding polymorphisms that may affect spatial and temporal regulation of gene expression.
Contrary to what is expected in Mendelian traits, subtle changes are postulated to influence the phenotypic outcome of complex (common) traits. Further functional studies may aid in identification of functional polymorphisms that may affect functional TFBS, which in turn may be used to uncover genetic regulatory interactions underlying normal cognitive variation.