Breakpoint mapping by next generation sequencing reveals causative gene disruption in patients carrying apparently balanced chromosome rearrangements with intellectual deficiency and/or congenital malformations
Breakpoint mapping by next generation sequencing reveals causative gene disruption in patients carrying apparently balanced chromosome rearrangements with intellectual deficiency and/or congenital malformations
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DOI：
10.1136/jmedgenet-2012-101351
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发表时间：
2013-03-01
影响因子：
4
通讯作者：
Sanlaville, Damien
中科院分区：
文献类型：
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作者：
Schluth-Bolard, Caroline;Labalme, Audrey;Sanlaville, Damien
背景在6%的病例中，明显平衡的染色体重排(ABCR)与异常表型相关。这可能是由于隐蔽的基因组不平衡或基因在断裂点的破坏。然而，使用常规方法(如荧光原位杂交(FISH)、Southern杂交)进行断点克隆往往费时费力。在这项工作中，我们使用下一代测序(NGS)在分子水平上定位了4例携带ABCR(1例易位、1例复杂染色体重排和2例倒位)的多发性先天性异常和/或智能缺陷(MCA/ID)患者的断裂点，对应于9个断裂点。采用全基因组配对末端分析方法识别断裂点。FISH和聚合酶链式反应结合Sanger测序对结果进行了验证。NGS发现了一个额外的断点，这是由于一位患者断点连接处的隐秘反转。10个断裂点中有9个发生在重复元件上，5个基因的内含子序列(TCF4、SHANK2、PPFIA1、RAB19、KCNQ1)发生突变。结论NGS是从分子水平快速克隆ABCR的有力工具。我们发现，在四分之三的患者中，基因中断可以解释表型，允许适应的遗传咨询和停止不必要的调查。我们建议，一旦通过阵列CGH排除了基因组失衡，携带ABCR的表型异常的患者应该进行NGS系统研究。
Background Apparently balanced chromosomal rearrangements (ABCR) are associated with an abnormal phenotype in 6% of cases. This may be due to cryptic genomic imbalances or to the disruption of genes at the breakpoint. However, breakpoint cloning using conventional methods (ie, fluorescent in situ hybridisation (FISH), Southern blot) is often laborious and time consuming. In this work, we used next generation sequencing (NGS) to locate breakpoints at the molecular level in four patients with multiple congenital abnormalities and/or intellectual deficiency (MCA/ID) who were carrying ABCR (one translocation, one complex chromosomal rearrangement and two inversions), which corresponded to nine breakpoints.Methods Genomic imbalance was previously excluded by array comparative genomic hybridisation (CGH) in all four patients. Whole genome paired-end protocol was used to identify breakpoints. The results were verified by FISH and by PCR with Sanger sequencing.Results We were able to map all nine breakpoints. NGS revealed an additional breakpoint due to a cryptic inversion at a breakpoint junction in one patient. Nine of 10 breakpoints occurred in repetitive elements and five genes were disrupted in their intronic sequence (TCF4, SHANK2, PPFIA1, RAB19, KCNQ1).Conclusions NGS is a powerful tool allowing rapid breakpoint cloning of ABCR at the molecular level. We showed that in three out of four patients, gene disruption could account for the phenotype, allowing adapted genetic counselling and stopping unnecessary investigations. We propose that patients carrying ABCR with an abnormal phenotype should be explored systematically by NGS once a genomic imbalance has been excluded by array CGH.