Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
J Invest Dermatol. 2011 Feb;131(2):480-6. doi: 10.1038/jid.2010.331. Epub 2010 Nov 18.
The presence of recurrent high-risk mutations in cyclin-dependent kinase inhibitor 2A/cyclin-dependent kinase 4 (CDKN2A/CDK4) among melanoma-prone families suggests that a high-throughput, multiplex assay could serve as an effective initial screening tool. To this end, we have developed a multiplex bead-based assay for high-throughput CDKN2A/CDK4 genotyping in the context of familial melanoma. Genomic DNA from 1,603 subjects (1,005 in training set and 598 in validation set) were amplified by multiplex PCR using five CDKN2A/CDK4 primer sets followed by multiplex allele-specific primer extension for 39 distinct germline variants. The products were then sorted and analyzed using the Luminex xMAP system. Genotypes were compared with previously determined sequence data. In the Toronto training cohort, all 145 samples with known variants were detected by the bead assay (100% concordance). Analysis of the 598 samples from the GenoMEL validation set led to identification of 150/155 expected variants (96.77%). Overall, the bead assay correctly genotyped 1,540/1,603 (96.07%) of all individuals in the study and 1,540/1,545 (99.68%) of individuals whose variants were represented in the probe set. Out of a total of 62,517 allelic calls, 62,512 (99.99%) were correctly assigned. The multiplex bead-based assay is an accurate method for genotyping CDKN2A/CDK4 variants and is potentially useful in genotyping low-to-moderate melanoma risk single-nucleotide polymorphisms.
在易患黑色素瘤的家族中，细胞周期蛋白依赖性激酶抑制剂 2A/细胞周期蛋白依赖性激酶 4（CDKN2A/CDK4）中反复出现的高危突变的存在表明，高通量、多重检测可以作为一种有效的初始筛选工具。为此，我们开发了一种用于家族性黑色素瘤中高通量 CDKN2A/CDK4 基因分型的多重珠基检测方法。使用五个 CDKN2A/CDK4 引物组对来自 1603 个个体（训练组 1005 个，验证组 598 个）的基因组 DNA 进行多重 PCR 扩增，然后对 39 种不同的种系变体进行多重等位基因特异性引物延伸。然后使用 Luminex xMAP 系统对产物进行分类和分析。将基因型与先前确定的序列数据进行比较。在多伦多训练队列中，珠粒检测法检测到所有 145 个已知变体的样本（100%一致性）。对 GenoMEL 验证组的 598 个样本的分析导致鉴定出 150/155 个预期变体（96.77%）。总体而言，珠粒检测法正确地对研究中所有个体的 1540/1603 个（96.07%）和个体的 1540/1545 个（99.68%）进行了基因分型，这些个体的变体包含在探针组中。在总共 62517 个等位基因调用中，正确分配了 62512 个（99.99%）。这种多重珠粒检测法是一种用于 CDKN2A/CDK4 变体基因分型的准确方法，在对低至中度黑色素瘤风险单核苷酸多态性进行基因分型方面具有潜在的应用价值。
