Enhancer Chip: Detecting Human Copy Number Variations in Regulatory Elements

Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.

人类基因组的非编码区域承载着关键的功能特性。近期多项成功研究表明，远端作用基因增强子序列的变异可能引发疾病。事实上，地中海贫血、轴前多指（趾）症以及先天性巨结肠易感性等多种病症，均可能由增强子元件的重排所导致。我们分析了基因组中增强子位点的分布情况，并将其定位与此前已报道的拷贝数变异（copy-number variations, CNVs）进行了比对。上述数据显示，拷贝数可变的增强子受到了负选择。为识别覆盖增强子元件的拷贝数变异，我们开发了一种简便且经济高效的检测方法。本文介绍了一款定制化寡核苷酸阵列比较基因组杂交（array-based comparative genomic hybridization, aCGH）芯片的基因筛选、设计策略与实验验证流程，该芯片被命名为“增强子芯片（Enhancer Chip）”。该芯片旨在用于拷贝数变异检测，可实现全基因组300kb分辨率的分析，并以十倍于该分辨率的精度检测特定疾病区域（端粒、着丝粒以及选定的疾病位点）。此外，这是首款能够检测超过1250个增强子的阵列比较基因组杂交芯片，可用于探究增强子的潜在致病作用。验证实验结果表明，“增强子芯片”可高效检测覆盖增强子位点的拷贝数重复与缺失，证明其是检测并解析拷贝数可变增强子的有力工具。