Münch Claudia, Kirsch Stefan, Fernandes António M G, Schempp Werner
Institute of Human Genetics and Anthropology, University of Freiburg, Breisacher Str, 33, 79106 Freiburg, Germany.
BMC Evol Biol. 2008 Oct 2;8:269. doi: 10.1186/1471-2148-8-269.
Segmental duplications (SDs) are euchromatic portions of genomic DNA (> or = 1 kb) that occur at more than one site within the genome, and typically share a high level of sequence identity (>90%). Approximately 5% of the human genome is composed of such duplicated sequences. Here we report the detailed investigation of CHEK2 duplications. CHEK2 is a multiorgan cancer susceptibility gene encoding a cell cycle checkpoint kinase acting in the DNA-damage response signalling pathway. The continuous presence of the CHEK2 gene in all eukaryotes and its important role in maintaining genome stability prompted us to investigate the duplicative evolution and phylogeny of CHEK2 and its paralogs during anthropoid evolution.
To study CHEK2 duplicon evolution in anthropoids we applied a combination of comparative FISH and in silico analyses. Our comparative FISH results with a CHEK2 fosmid probe revealed the single-copy status of CHEK2 in New World monkeys, Old World monkeys and gibbons. Whereas a single CHEK2 duplication was detected in orangutan, a multi-site signal pattern indicated a burst of duplication in African great apes and human. Phylogenetic analysis of paralogous and ancestral CHEK2 sequences in human, chimpanzee and rhesus macaque confirmed this burst of duplication, which occurred after the radiation of orangutan and African great apes. In addition, we used inter-species quantitative PCR to determine CHEK2 copy numbers. An amplification of CHEK2 was detected in African great apes and the highest CHEK2 copy number of all analysed species was observed in the human genome. Furthermore, we detected variation in CHEK2 copy numbers within the analysed set of human samples.
Our detailed analysis revealed the highly dynamic nature of CHEK2 duplication during anthropoid evolution. We determined a burst of CHEK2 duplication after the radiation of orangutan and African great apes and identified the highest CHEK2 copy number in human. In conclusion, our analysis of CHEK2 duplicon evolution revealed that SDs contribute to inter-species variation. Furthermore, our qPCR analysis led us to presume CHEK2 copy number variation in human, and molecular diagnostics of the cancer susceptibility gene CHEK2 inside the duplicated region might be hampered by the individual-specific set of duplicons.
片段重复（SDs）是基因组DNA的常染色质部分（≥1 kb），在基因组内的多个位点出现，并且通常具有高水平的序列同一性（>90%）。人类基因组约5%由这类重复序列组成。在此，我们报告对CHEK2重复的详细研究。CHEK2是一种多器官癌症易感基因，编码一种在DNA损伤反应信号通路中起作用的细胞周期检查点激酶。CHEK2基因在所有真核生物中的持续存在及其在维持基因组稳定性中的重要作用促使我们研究在类人猿进化过程中CHEK2及其旁系同源基因的重复进化和系统发育。
为了研究类人猿中CHEK2复制子的进化，我们应用了比较荧光原位杂交（FISH）和计算机分析相结合的方法。我们用CHEK2 fosmid探针进行的比较FISH结果显示，新世界猴、旧世界猴和长臂猿中的CHEK2呈单拷贝状态。而在猩猩中检测到一次CHEK2重复，多位点信号模式表明非洲大猿和人类中出现了重复爆发。对人类、黑猩猩和恒河猴中旁系同源和祖先CHEK2序列的系统发育分析证实了这次重复爆发，其发生在猩猩和非洲大猿分化之后。此外，我们使用种间定量PCR来确定CHEK2的拷贝数。在非洲大猿中检测到CHEK2的扩增，并且在人类基因组中观察到所有分析物种中最高的CHEK2拷贝数。此外，我们在分析的人类样本集中检测到CHEK2拷贝数的变异。
我们的详细分析揭示了类人猿进化过程中CHEK2重复的高度动态性质。我们确定在猩猩和非洲大猿分化之后出现了CHEK2重复爆发，并在人类中确定了最高的CHEK2拷贝数。总之，我们对CHEK2复制子进化的分析表明，SDs导致了种间变异。此外，我们的qPCR分析使我们推测人类中存在CHEK2拷贝数变异，并认为重复区域内癌症易感基因CHEK2的分子诊断可能会受到个体特异性复制子集的阻碍。
