de Lima Leonardo Gomes, Howe Edmund, Singh Vijay Pratap, Potapova Tamara, Li Hua, Xu Baoshan, Castle Jemma, Crozier Steve, Harrison Christine J, Clifford Steve C, Miga Karen H, Ryan Sarra L, Gerton Jennifer L
The Stowers Institute for Medical Research, Kansas City, MO, USA.
Lead contact.
Cell Genom. 2021 Dec;1(3). doi: 10.1016/j.xgen.2021.100064. Epub 2021 Dec 8.
Centromeric α-satellite repeats represent ~6% of the human genome, but their length and repetitive nature make sequencing and analysis of those regions challenging. However, centromeres are essential for the stable propagation of chromosomes, so tools are urgently needed to monitor centromere copy number and how it influences chromosome transmission and genome stability. We developed and benchmarked droplet digital PCR (ddPCR) assays that measure copy number for five human centromeric arrays. We applied them to characterize natural variation in centromeric array size, analyzing normal tissue from 37 individuals from China and 39 individuals from the US and UK. Each chromosome-specific array varies in size up to 10-fold across individuals and up to 50-fold across chromosomes, indicating a unique complement of arrays in each individual. We also used the ddPCR assays to analyze centromere copy number in 76 matched tumor-normal samples across four cancer types, representing the most-comprehensive quantitative analysis of centromeric array stability in cancer to date. In contrast to stable transmission in cultured cells, centromeric arrays show gain and loss events in each of the cancer types, suggesting centromeric α-satellite DNA represents a new category of genome instability in cancer. Our methodology for measuring human centromeric-array copy number will advance research on centromeres and genome integrity in normal and disease states.
着丝粒α卫星重复序列约占人类基因组的6%，但其长度和重复性质使得对这些区域进行测序和分析具有挑战性。然而，着丝粒对于染色体的稳定传递至关重要，因此迫切需要工具来监测着丝粒拷贝数及其对染色体传递和基因组稳定性的影响。我们开发并验证了用于测量五个人类着丝粒阵列拷贝数的液滴数字PCR（ddPCR）检测方法。我们将其应用于表征着丝粒阵列大小的自然变异，分析了来自中国的37名个体以及来自美国和英国的39名个体的正常组织。每个染色体特异性阵列在个体间大小变化可达10倍，在不同染色体间可达50倍，这表明每个个体的阵列组合都是独特的。我们还使用ddPCR检测方法分析了四种癌症类型中76对匹配的肿瘤-正常样本的着丝粒拷贝数，这是迄今为止对癌症中着丝粒阵列稳定性最全面的定量分析。与培养细胞中的稳定传递不同，着丝粒阵列在每种癌症类型中都显示出增减事件，这表明着丝粒α卫星DNA代表了癌症中基因组不稳定的一种新类型。我们测量人类着丝粒阵列拷贝数的方法将推动对正常和疾病状态下着丝粒及基因组完整性的研究。
