Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
Neurobiol Aging. 2012 Aug;33(8):1848.e1-13. doi: 10.1016/j.neurobiolaging.2012.02.005. Epub 2012 Mar 8.
Nicastrin (NCSTN) is a component of the γ-secretase complex and therefore potentially a candidate risk gene for Alzheimer's disease. Here, we have developed a novel functional genomics methodology to express common locus haplotypes to assess functional differences. DNA recombination was used to engineer 5 bacterial artificial chromosomes (BACs) to each express a different haplotype of the NCSTN locus. Each NCSTN-BAC was delivered to knockout nicastrin (Ncstn(-/-)) cells and clonal NCSTN-BAC(+)/Ncstn(-/-) cell lines were created for functional analyses. We showed that all NCSTN-BAC haplotypes expressed nicastrin protein and rescued γ-secretase activity and amyloid beta (Aβ) production in NCSTN-BAC(+)/Ncstn(-/-) lines. We then showed that genetic variation at the NCSTN locus affected alternative splicing in human postmortem brain tissue. However, there was no robust functional difference between clonal cell lines rescued by each of the 5 different haplotypes. Finally, there was no statistically significant association of NCSTN with disease risk in the 4 cohorts. We therefore conclude that it is unlikely that common variation at the NCSTN locus is a risk factor for Alzheimer's disease.
尼卡斯特林（NCSTN）是γ-分泌酶复合物的一个组成部分，因此可能是阿尔茨海默病的候选风险基因。在这里，我们开发了一种新的功能基因组学方法来表达常见的基因座单体型，以评估功能差异。利用 DNA 重组技术构建了 5 个细菌人工染色体（BAC），每个 BAC 表达 NCSTN 基因座的不同单体型。将每个 NCSTN-BAC 递送到敲除尼卡斯特林（Ncstn(-/-)）细胞中，并创建了具有 NCSTN-BAC(+)/Ncstn(-/-)克隆的细胞系，以进行功能分析。我们表明，所有 NCSTN-BAC 单体型都表达了尼卡斯特林蛋白，并在 NCSTN-BAC(+)/Ncstn(-/-) 细胞系中恢复了 γ-分泌酶活性和淀粉样β（Aβ）产生。然后，我们表明 NCSTN 基因座的遗传变异影响了人类尸检脑组织中的选择性剪接。然而，在由 5 个不同单体型中的每一个拯救的克隆细胞系之间没有明显的功能差异。最后，在 4 个队列中，NCSTN 与疾病风险之间没有统计学上的显著关联。因此，我们得出结论，NCSTN 基因座的常见变异不太可能是阿尔茨海默病的风险因素。
