Department of Psychiatry and Neurobehavioral Sciences, University of Virginia, Charlottesville, VA, USA; Shanxi Key Laboratory of Environmental Veterinary Sciences, Shanxi Agricultural University, Shanxi, China.
Neurosci Lett. 2013 Oct 25;555:215-9. doi: 10.1016/j.neulet.2013.09.040. Epub 2013 Sep 26.
Recently, we reported that several polymorphisms and haplotypes in the choline acetyltransferase gene (ChAT) are associated with nicotine dependence (ND). Of them, SNP rs1880676 is of particular interest because: (1) it is a non-synonymous variant located in the coding region of an alternatively spliced form of ChAT and (2) it is located in several haplotypes that are significantly associated with ND. The objective of this study was to determine, using an in vitro system, whether the alleles G (coding for aspartic acid) or A (coding for asparagine) of rs1880676 have any allele-specific effect on ChAT expression. We first used site-directed mutagenesis to construct two expression vectors differed in the allelic position of rs1880676 (G/A), which were transfected into HeLa cells. We then measured expression of ChAT associated with each allele. We found significant expression differences for the two alleles, with the G allele being expressed significantly greater than A allele (P<0.01 at both mRNA and protein levels). Further, we validated the ChAT expression of the G allele was significantly higher than that of the A allele by using ELISA assay (P=0.00016). We concluded that rs1880676 is functional and that the allelic variations of this polymorphism are involved in developing ND by altering ChAT expression.
最近，我们报道了胆碱乙酰转移酶基因（ChAT）中的几个多态性和单倍型与尼古丁依赖（ND）有关。其中，SNP rs1880676 特别引人注目，因为：（1）它是一个非同义突变，位于 ChAT 一种选择性剪接形式的编码区；（2）它位于与 ND 显著相关的几个单倍型中。本研究的目的是使用体外系统确定 rs1880676 的等位基因 G（编码天冬氨酸）或 A（编码天冬酰胺）是否对 ChAT 表达有任何等位基因特异性影响。我们首先使用定点诱变构建了两个在 rs1880676 等位基因位置（G/A）上不同的表达载体，将其转染到 HeLa 细胞中。然后，我们测量了与每个等位基因相关的 ChAT 表达。我们发现两个等位基因的表达存在显著差异，G 等位基因的表达显著高于 A 等位基因（mRNA 和蛋白质水平的 P<0.01）。此外，我们通过 ELISA 测定进一步验证了 G 等位基因的 ChAT 表达明显高于 A 等位基因（P=0.00016）。我们得出结论，rs1880676 是功能性的，该多态性的等位基因变异通过改变 ChAT 表达参与了 ND 的发生。
