Department of Pathology, Molecular Pathology Laboratory, Christian Medical College, Vellore, 632004, Tamil Nadu, India.
Fam Cancer. 2013 Sep;12(3):519-24. doi: 10.1007/s10689-013-9606-2.
Mutation in VHL gene causes the von Hippel-Lindau (VHL) disease, a dominantly inherited familial cancer syndrome. The VHL mutation pattern includes point mutations, small deletions and large deletions. While most mutations can be identified during sequencing, large deletions often remain unnoticed in initial mutational screening. We evaluated the utility of a previously described real-time quantitative PCR (RQ-PCR) using SYBR Green for detection of larger deletions in the VHL gene and normalized the data using two reference genes with a normal copy number i.e., ZNF80 (3q13.31) and GPR15 (3q12.1). DNA sequencing was also done on all cases included in the study. SJNB-6 cell line demonstrating distal 3p loss was used as a positive control for deletion. Out of 21 individual cases included of VHL disease, 2 cases were found with partial deletion by RQ-PCR, with an exon 1 deletion, while PCR-sequencing identified 5 cases with base pair substitution and 1 with splice site variant which were not picked up by RQ-PCR. RQ-PCR proved to be fast, accurate and sensitive for identifying large deletions and can be incorporated into the routine work-up for detection of large deletions in VHL disease.
VHL 基因突变导致 von Hippel-Lindau（VHL）病，这是一种显性遗传家族性癌症综合征。VHL 基因突变模式包括点突变、小缺失和大片段缺失。虽然大多数突变可以在测序过程中被识别，但大片段缺失在初始突变筛选中往往被忽略。我们评估了先前描述的使用 SYBR Green 的实时定量 PCR（RQ-PCR）检测 VHL 基因中较大缺失的效用，并使用两个正常拷贝数的参考基因（即 ZNF80（3q13.31）和 GPR15（3q12.1））对数据进行归一化。所有纳入研究的病例均进行 DNA 测序。SJNB-6 细胞系表现出远端 3p 缺失，用作缺失的阳性对照。在 21 例 VHL 病个体病例中，有 2 例通过 RQ-PCR 发现部分缺失，存在外显子 1 缺失，而 PCR 测序鉴定出 5 例碱基对取代和 1 例剪接位点变异，这些变异未被 RQ-PCR 检出。RQ-PCR 被证明可快速、准确和敏感地识别大片段缺失，并可纳入 VHL 病中大片段缺失检测的常规工作流程。
