Pollock P M, Welch J, Hayward N K
Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892-4094, USA.
Cancer Res. 2001 Feb 1;61(3):1154-61.
Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 9p involved in the development of melanoma. Although LOH at 9p has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 9p. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations by single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele. Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748 the markers closest to CDKN2A. Of the remaining 11 tumors with LOH 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion. This report supports the conclusions of previous studies that a least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.
细胞遗传学和杂合性缺失（LOH）研究长期以来表明，9号染色体短臂上存在一个与黑色素瘤发生发展相关的肿瘤抑制基因（TSG）。尽管在大约60%的黑色素瘤肿瘤中报道了9p处的杂合性缺失，但这些肿瘤中只有5 - 10%被证明携带CDKN2A突变，这增加了另一个参与黑色素瘤的TSG定位于9号染色体短臂的可能性。为了研究这种可能性，通过单链构象多态性分析和测序对来自35个个体的37例黑色素瘤进行了CDKN2A突变分析。然后对黑色素瘤样本进行了15个定位于9p13 - 24的标记物分型，以研究该区域的LOH趋势。在那些在CDKN2A侧翼标记物处显示杂合性保留且在基因一侧或两侧存在LOH的肿瘤中，进行了多重微卫星PCR以排除包含CDKN2A区域的纯合缺失。在5例患者的肿瘤中发现了CDKN2A突变[35例中的5例（14%）]，其中4例在整个检测区域均显示杂合性缺失。其余未观察到杂合性缺失的肿瘤携带两个点突变，每个等位基因各一个。尽管在37例黑色素瘤肿瘤中的22例（59%）对应35个个体中的20例（57%）的一个或多个标记物处鉴定出了杂合性缺失，但只有来自9个个体的11例肿瘤[35例中的9例（26%）]在最接近CDKN2A的标记物D9S942和D9S1748处显示杂合性缺失。在其余11例存在杂合性缺失的肿瘤中，9例在CDKN2A着丝粒和/或端粒方向的两个或更多相邻标记物处显示杂合性缺失，而在与CDKN2A相邻的几个标记物处保留杂合性。多重PCR显示一个肿瘤携带从D9S1748延伸至IFN-α基因座的纯合缺失。在其余8例肿瘤中，多重PCR表明观察到的杂合性并非归因于D9S1748、D9S942或D9S974处的纯合缺失和基质污染，通过比较扩增强度来衡量，这表明侧翼杂合性缺失时杂合性保留并不总是表明存在纯合缺失。本报告支持先前研究的结论，即除CDKN2A外，至少还有两个参与黑色素瘤发生发展的TSG可能位于9号染色体短臂上。
