Unit of Molecular Mutagenesis and DNA Repair, National Institute for Cancer Research IST, 16132 Genoa, Italy.
Hum Mol Genet. 2010 Apr 15;19(8):1479-91. doi: 10.1093/hmg/ddq022. Epub 2010 Jan 21.
Germline CDKN2A mutations are observed in 20-50% of melanoma-prone families. We identified melanoma patients that were heterozygous for non-coding germline variants in the 5'-UTR of CDKN2A (c.-21C > T; c.-25C > T&c.-180G > A; c.-56G > T; c.-67G > C) and examined their impact on the p16(INK4a) 5'-UTR activity using two luciferase-based reporter vectors that differ in basal transcription level and that were transfected into the melanoma-derived WM266-4 and in the breast cancer-derived MCF7 cells. The wild-type 5'-UTR sequence, containing a reported SNP (c.-33G > C) and a known melanoma-predisposing mutation (c.-34G > T), was included as controls. Results revealed that the variants at -21 and -34 severely reduced the reporter activity. The variants at -56 and at -25&-180 exhibited a milder impact, while results with c.-67G > C were dependent on the plasmid type. Quantification of the luciferase mRNA indicated that the effects of the variants were mainly post-transcriptional. Using a bicistronic dual-luciferase reporter plasmid, we confirmed that c.-21C > T and c.-34G > T had a severe negative impact in both cell lines. We also applied a polysomal profiling technique to samples heterozygous for the 5'-UTR variants, including patient-derived lymphoblasts. Analysis of allelic imbalance indicated that in addition to the c.-21C > T variant, the c.-56T > G and c.-67G > C variants also reduced mRNA translation efficiency. Overall, our results suggest that the c.-21C > T sequence variant is a melanoma-predisposing mutation. The c.-25C > T&c.-180G > A and particularly the c.-56G > T variants showed a range of intermediate functional defects in the different assays, and were not observed in the control population. We propose that these variants should be considered as potential mutations.
胚系 CDKN2A 突变在 20-50%的黑色素瘤易感家族中被观察到。我们鉴定了黑色素瘤患者，他们在 CDKN2A 的 5'-UTR 中存在非编码胚系变体的杂合性（c.-21C>T；c.-25C>T 和 c.-180G> A；c.-56G>T；c.-67G>C），并使用两种基于荧光素酶的报告载体检测它们对 p16(INK4a)5'-UTR 活性的影响，这两种报告载体在基础转录水平上存在差异，并转染到黑色素瘤衍生的 WM266-4 和乳腺癌衍生的 MCF7 细胞中。野生型 5'-UTR 序列包含报道的 SNP（c.-33G>C）和已知的黑色素瘤易感突变（c.-34G>T），作为对照。结果表明，-21 和-34 处的变体严重降低了报告基因的活性。-56 和-25&-180 处的变体表现出较轻的影响，而 c.-67G>C 的结果依赖于质粒类型。荧光素酶 mRNA 的定量表明，变体的影响主要是转录后。使用双顺反子双荧光素酶报告质粒，我们在两种细胞系中证实了 c.-21C>T 和 c.-34G>T 具有严重的负向影响。我们还应用多核糖体谱技术对包括患者衍生的淋巴母细胞在内的 5'-UTR 变体杂合子进行了样本分析。等位基因失衡分析表明，除了 c.-21C>T 变体外，c.-56T>G 和 c.-67G>C 变体也降低了 mRNA 翻译效率。总体而言，我们的结果表明 c.-21C>T 序列变体是一种黑色素瘤易感突变。c.-25C>T&c.-180G>A 特别是 c.-56G>T 变体在不同的检测中表现出一系列中间功能缺陷，并且在对照人群中未观察到。我们建议将这些变体视为潜在的突变。
