Hata Nobuhiro, Yoshimoto Koji, Yokoyama Nobuhiko, Mizoguchi Masahiro, Shono Tadahisa, Guan Yanlei, Tahira Tomoko, Kukita Yoji, Higasa Koichiro, Nagata Shinji, Iwaki Toru, Sasaki Tomio, Hayashi Kenshi
Department of Neurosurgery, Graduate School of Medical Sciences, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Clin Chem. 2006 Mar;52(3):370-8. doi: 10.1373/clinchem.2005.060954. Epub 2006 Jan 5.
Detection of loss of heterozygosity (LOH) in clinical tissue samples is frequently difficult because samples are usually contaminated with noncancerous cells or because tumor cells in single tissues have genetic heterogeneity, and the precision of available techniques is insufficient for reliable analysis in such materials. We hypothesized that single-strand conformation polymorphism (SSCP) analysis can precisely quantify the gene dosage in mixed samples and is suitable for detection of LOH in clinical tissue samples.
We assessed the accuracy of a fluorescent SSCP method for the quantification of single-nucleotide polymorphism (SNP) alleles, using DNAs that were composed of cancerous DNA mixed with noncancerous DNA at various ratios. We applied this method to precisely characterize LOH in glioma tissue samples, using 96 SNPs that were evenly distributed throughout chromosome 10.
LOH could be detected even in the cancerous DNA heavily contaminated (up to 80%) with noncancerous DNA. Using this method, we obtained LOH profiles of 56 gliomas with resolution at the SNP level (i.e., 1.5-Mbp interval). Anaplastic astrocytomas exhibited both 10p and 10q LOH, whereas diffuse astrocytomas frequently (63% of the cases) exhibited loss of 10p alone. We also found a possible new LOH region (around 10p13) in gliomas.
The present method is effective for precise mapping of LOH region in surgically obtained tumor tissues and could be applicable to the genetic diagnosis of cancers other than gliomas.
在临床组织样本中检测杂合性缺失（LOH）通常很困难，因为样本通常被非癌细胞污染，或者因为单个组织中的肿瘤细胞具有基因异质性，而且现有技术的精度不足以对这类材料进行可靠分析。我们假设单链构象多态性（SSCP）分析可以精确量化混合样本中的基因剂量，并且适用于检测临床组织样本中的LOH。
我们使用以不同比例混合癌细胞DNA和非癌细胞DNA的DNA，评估了荧光SSCP方法对单核苷酸多态性（SNP）等位基因定量的准确性。我们应用该方法，利用均匀分布在10号染色体上的96个SNP，精确表征胶质瘤组织样本中的LOH。
即使在被非癌细胞DNA严重污染（高达80%）的癌细胞DNA中也能检测到LOH。使用这种方法，我们获得了56例胶质瘤的LOH图谱，分辨率达到SNP水平（即1.5-Mbp间隔）。间变性星形细胞瘤同时表现出10p和10q LOH，而弥漫性星形细胞瘤经常（63%的病例）仅表现出10p缺失。我们还在胶质瘤中发现了一个可能的新LOH区域（约10p13附近）。
本方法对于手术获得的肿瘤组织中LOH区域的精确图谱绘制是有效的，并且可能适用于胶质瘤以外的其他癌症的基因诊断。
