Hemoglobinopathies Laboratory, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Hum Mutat. 2012 Jan;33(1):272-80. doi: 10.1002/humu.21612. Epub 2011 Oct 31.
Implementation of multiplex ligation-dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap-PCRs. We have designed a custom fine-tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high-resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency.
多重连接依赖探针扩增（MLPA）技术用于检测导致地中海贫血的缺失，从而发现新的重排。了解确切的断点序列可以更深入地了解这些重排的分子机制，并有助于设计缺口-PCR。我们设计了一个带有覆盖完整珠蛋白基因簇的寡核苷酸的定制精细镶嵌阵列。我们杂交了 27 个含有新鉴定缺失的 DNA 样本和 9 个阳性对照。我们设计了特定的引物来扩增包含断点序列的相对较短的片段，并通过直接测序进行分析。9 个阳性对照的结果表明，阵列比较基因组杂交（aCGH）适用于检测小的和大的重排。我们能够将所有的断点定位到大约 2 kb 的区域。我们为 22 个病例设计了断点引物，其中 19 个病例的扩增成功。对于其中的 12 个病例，确定了断点的确切位置。其中 7 个缺失以前没有报道过。aCGH 是一种用于高分辨率断点特征分析的有价值的工具。MLPA 和 aCGH 的结合导致了相对便宜且易于执行的 PCR 检测，对于只有少数特定缺失以高频率发生的人群中的实验室来说，它可能是 MLPA 的替代方法。
