Haplotyping of putative microRNA‐binding sites in the SNAP‐25 gene

Abstract

Synaptosomal-associated protein 25 (SNAP-25) plays a crucial role in exocitosis. Single nucleotide polymorphisms (rs3746544 and rs1051312) in the 3' un-translated region of the SNAP-25 gene have been described to be in association with attention-deficit hyperactivity disorder. As the disease affects millions of children world-wide, understanding the genetic background of attention-deficit hyperactivity disorder is of crucial importance. Efficient and reliable PCR-RFLP protocols were elaborated for the genotyping of the rs3746544 and rs1051312 SNPs employing a high-throughput capillary electrophoresis method for fragment analysis. A novel real-time PCR-based technique was used applying sequence specific TaqMan probes to haplotype the two SNPs, and the G-C haplotype could not be detected in a large Caucasian population (N=1376). These findings have been confirmed by molecular biology tools as well as by the PHASE Bayesian computational approach. In silico analyses have suggested that the two SNPs might alter microRNA binding and thus have an effect on SNAP-25 production. We have demonstrated that this biological information can be revealed only by direct haplotype analysis emphasizing the importance of our novel molecular haplotye analysis protocol. Results of the study of the two SNPs might shed light on the association of SNAP-25 variants and pathological phenotypes at the molecular level.

Association of Impulsivity and Polymorphic MicroRNA-641 Target Sites in the SNAP-25 Gene

“…On the other hand, the two SNPs of the promoter region were genotyped individually as their greater distance did not allow a direct haplotype analysis, and haplotype frequencies were estimated by calculation. Analysis of the 3′ UTR haplotypes confirmed our previous result [19], that the G–C haplotype did not occur at all in the population. Interestingly a similar situation was observed regarding the promoter haplotypes.…”

“…Taking into consideration that our study investigated impulsivity instead of ADHD in a healthy population, all data support, that SNAP-25 is one of the several genetic factors of impulsivity and related psychiatric disorders. Interestingly the haplotype with double mismatch (G–C) in the miRNA target site of SNAP-25 gene was completely undetectable in our healthy population of European descent (Hungarian) according to our previous [19] and current studies. It is important to note that haplotypes were not estimated but individually measured by a direct molecular haplotype analysis method developed earlier [19], [41].…”

“…Interestingly the haplotype with double mismatch (G–C) in the miRNA target site of SNAP-25 gene was completely undetectable in our healthy population of European descent (Hungarian) according to our previous [19] and current studies. It is important to note that haplotypes were not estimated but individually measured by a direct molecular haplotype analysis method developed earlier [19], [41]. A theoretically possible explanation of the absence of the G–C haplotype in our healthy volunteer population could be the more severe effect of this haplotype on SNAP-25 gene expression leading to pathophysiological consequences.…”

“…As the two SNPs (rs3746544 and rs1051312) in the 3′ UTR are separated by only 3 basepairs from each other, it was possible to identify haplotypes directly in each individual sample using the published “double-tube” method [19]. In summary, the method is based on the application of haplotype-specific TaqMan probes in a real-time polymerase chain reaction.…”

“…There are two SNPs (rs3746544 and rs1051312) in the 3′ UTR of the SNAP-25 gene with special interest, we previously demonstrated by in silico sequence analysis that they are localized in the putative target site of miR-641 [19].…”