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首页> 外文期刊>Molecular cytogenetics >Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes
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Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes

机译：设计和验证用于改善超数字标记染色体的诊断和表型预测的着丝粒BAC克隆集

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Background Small supernumerary marker chromosomes (sSMCs) are additional, structurally abnormal chromosomes, generally smaller than chromosome 20 of the same metaphase spread. Due to their small size, they are difficult to characterize by conventional cytogenetics alone. In regard to their clinical effects, sSMCs are a heterogeneous group: in particular, sSMCs containing pericentromeric euchromatin are likely to be associated with abnormal outcomes, although exceptions have been reported. To improve characterization of the genetic content of sSMCs, several approaches might be applied based on different molecular and molecular-cytogenetic assays, e.g., fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (array CGH), and multiplex ligation-dependent probe amplification (MLPA). To provide a complementary tool for the characterization of sSMCs, we constructed and validated a new, FISH-based, pericentromeric Bacterial Artificial Chromosome (BAC) clone set that with a high resolution spans the most proximal euchromatic sequences of all human chromosome arms, excluding the acrocentric short arms. Results By FISH analysis, we assayed 561 pericentromeric BAC probes and excluded 75 that showed a wrong chromosomal localization. The remaining 486 probes were used to establish 43 BAC-based pericentromeric panels. Each panel consists of a core, which with a high resolution covers the most proximal euchromatic ~0.7 Mb (on average) of each chromosome arm and generally bridges the heterochromatin/euchromatin junction, as well as clones located proximally and distally to the core. The pericentromeric clone set was subsequently validated by the characterization of 19 sSMCs. Using the core probes, we could rapidly distinguish between heterochromatic (1/19) and euchromatic (11/19) sSMCs, and estimate the euchromatic DNA content, which ranged from approximately 0.13 to more than 10 Mb. The characterization was not completed for seven sSMCs due to a lack of information about the covered region in the reference sequence (1/19) or sample insufficiency (6/19). Conclusions Our results demonstrate that this pericentromeric clone set is useful as an alternative tool for sSMC characterization, primarily in cases of very small SMCs that contain either heterochromatin exclusively or a tiny amount of euchromatic sequence, and also in cases of low-level or cryptic mosaicism. The resulting data will foster knowledge of human proximal euchromatic regions involved in chromosomal imbalances, thereby improving genotype–phenotype correlations.
机译：背景技术小数量标记染色体（sSMCs）是其他结构异常的染色体，通常小于相同中期扩散染色体的20号染色体。由于它们的体积小，它们难以仅通过常规细胞遗传学来表征。就其临床效果而言，sSMCs是一个异质性的群体：特别是，尽管有报道报道例外，但含有周围着丝粒常染色质的sSMCs可能与异常结果相关。为了改善sSMC遗传成分的特征，可以基于不同的分子和分子细胞遗传学检测方法，例如荧光原位杂交（FISH），基于阵列的比较基因组杂交（阵列CGH）和多重连接-依赖性探针扩增（MLPA）。为了提供表征sSMC的补充工具，我们构建并验证了一种新的，基于FISH的，基于着丝粒的细菌人工染色体（BAC）克隆集，该克隆集具有较高的分辨率，涵盖了所有人类染色体臂的最接近的常染色体序列。近端短臂。结果通过FISH分析，我们分析了561个着丝粒BAC探针，并排除了75个显示错误的染色体定位的探针。其余的486个探针用于建立43个基于BAC的着丝粒组。每个面板由一个核心组成，该核心的高分辨率覆盖每个染色体臂的最近端常色〜0.7 Mb（平均），通常跨异染色质/常染色质连接点，以及位于核心近端和远端的克隆。随后通过19个sSMC的特征验证了着丝粒体克隆集。使用核心探针，我们可以快速区分异色（1/19）和常色（11/19）sSMC，并估计常色DNA含量，范围从大约0.13到10 Mb以上。由于缺少有关参考序列（1/19）或样本不足（6/19）中所覆盖区域的信息，七个sSMC的表征未完成。结论我们的结果表明，这种着丝粒克隆集可作为sSMC表征的替代工具，主要是在非常小的SMC中仅包含异染色质或少量常染色体序列的情况下，以及在低水平或隐秘镶嵌作用下。所得的数据将有助于人们了解与染色体失衡有关的人类近端常染色体区域，从而改善基因型与表型的相关性。

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