Vaandrager J W, Schuuring E, Zwikstra E, de Boer C J, Kleiverda K K, van Krieken J H, Kluin-Nelemans H C, van Ommen G J, Raap A K, Kluin P M
Department of Pathology, Leiden University Hospital, Leiden University, The Netherlands.
Blood. 1996 Aug 15;88(4):1177-82.
Several hematologic malignancies are associated with specific chromosomal translocations. Because of the dispersed distribution, chromosomal breakpoints may be difficult to detect using molecular techniques. We present a new application of a recently developed method, DNA fiber fluorescence in situ hybridization (fiber FISH), which allows direct visualization and mapping of chromosomal breakpoints. We tested this method for detection of the t(11;14)(q13;q32) translocation in mantle cell lymphoma. In DNA fiber FISH, a series of fluorochrome-labeled DNA probes covering several hundreds of kilobasepairs is hybridized to linear DNA molecules (or fibers) prepared from frozen tissue or intact cells. By using alternate fluorescent colors, a potential breakpoint region is stained in a color barcode pattern. Breaks in this region will split the barcode in two complementary parts, from which the breakpoint position can be derived. We used a 250-kb barcode covering the BCL-1 locus to detect 11q13 breakpoints in 20 well-characterized mantle cell lymphomas. A t(11;14) was shown by cohybridization of these probes with probes for the Ig heavy chain locus at 14q32. In 18 of 20 mantle cell lymphomas, a breakpoint within the 11q13/BCL-1 barcode was shown by the presence of multiple, complementary translocation products. Fusion of 11q13 and 14q32 sequences on single fibers indicating t(11;14)(q13;q32) was found in all 18 breakpoint-positive mantle cell lymphomas. In one additional case, fusion of an intact 11q13 barcode with 14q32 sequences indicated a breakpoint 100 kb centromeric of the major translocation cluster of BCL-1. Within the 120-kb region of BCL-1, breakpoints were widely scattered. This explains why, so far, a BCL-1 breakpoint had been detected by Southern blot analysis in only 10 of 19 cases. DNA fiber FISH analysis showed a t(11;14) in 95% of mantle cell lymphoma. The results indicate that DNA fiber FISH is a rapid, simple, and equally powerful method for detection of clustered and dispersed translocation breakpoints.
几种血液系统恶性肿瘤与特定的染色体易位相关。由于分布分散，使用分子技术可能难以检测到染色体断点。我们展示了一种最近开发的方法——DNA纤维荧光原位杂交（纤维FISH）的新应用，它可以直接可视化和定位染色体断点。我们测试了该方法用于检测套细胞淋巴瘤中的t(11;14)(q13;q32)易位。在DNA纤维FISH中，一系列覆盖数百千碱基对的荧光染料标记的DNA探针与从冷冻组织或完整细胞制备的线性DNA分子（或纤维）杂交。通过使用交替的荧光颜色，潜在的断点区域以彩色条码模式染色。该区域的断裂会将条码分成两个互补部分，由此可以确定断点位置。我们使用一个覆盖BCL-1基因座的250 kb条码来检测20例特征明确的套细胞淋巴瘤中的11q13断点。通过将这些探针与位于14q32的免疫球蛋白重链基因座的探针共杂交，显示出t(11;14)。在20例套细胞淋巴瘤中的18例中，11q13/BCL-1条码内的断点通过多个互补易位产物的存在得以显示。在所有18例断点阳性的套细胞淋巴瘤中均发现单纤维上11q13和14q32序列的融合，表明存在t(11;14)(q13;q32)。在另外1例中，完整的11q13条码与14q32序列的融合表明断点位于BCL-1主要易位簇着丝粒侧100 kb处。在BCL-1的120 kb区域内，断点广泛分散。这解释了为什么到目前为止，在19例病例中只有10例通过Southern印迹分析检测到BCL-1断点。DNA纤维FISH分析显示95%的套细胞淋巴瘤存在t(11;14)。结果表明，DNA纤维FISH是一种快速、简单且同样强大的方法，用于检测聚集和分散的易位断点。
