Laboratory of Molecular Diagnostics, Jeroen Bosch Hospital, 's-Hertogenbosch, The Netherlands.
PLoS One. 2012;7(7):e38362. doi: 10.1371/journal.pone.0038362. Epub 2012 Jul 2.
During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n=6 25-50% and n=6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25-50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci.
在肿瘤发展过程中，常发生杂合性丢失（LOH）。当 LOH 发生在前癌基因激活突变之后，如果野生型等位基因丢失或失活，突变等位基因可能会进一步增强。在骨髓增殖性肿瘤（MPN）中，JAK2V617F 的体细胞获得可能会导致 LOH，从而导致野生型等位基因丢失。LOH 在 MPN 和其他增殖性疾病中的发生可能会进一步增强突变等位基因，从而增加发病率。开发并验证了一种基于实时 PCR 的 SNP 分析方法，用于 JAK2 区域（JAK2LOH）的 LOH 检测。采集了 12 例 JAK2V617F 阳性患者（n=6，25-50%和 n=6，>50% JAK2V617F）和 81 例疑似 MPN 患者的 EDTA 保存血液，随后用于验证。为了生成种系谱，对每位患者的非肿瘤性福尔马林固定石蜡包埋组织进行了分析。SNP 分析的结果与已建立的短串联重复（STR）分析进行了比较。两种检测方法均在 6/6 例 25-50% JAK2V617F 的患者中检测到 JAK2LOH。在 JAK2V617F ＞50%的患者中，SNP 检测法在 6/6 例患者中检测到 JAK2LOH，STR 检测法在 5/6 例患者中检测到 JAK2LOH。在 81 例疑似 MPN 的患者中，有 18 例患者携带 JAK2V617F。SNP 和 STR 检测均显示其中 5 例存在 JAK2LOH。在 63 例 JAK2V617F 阴性患者中，SNP 和 STR 分析均未观察到 JAK2LOH。本研究提出的 SNP 检测方法可可靠地检测 JAK2LOH，是 STR 分析的快速、简便的替代方法。因此，我们预计 SNP 方法可作为开发用于其他临床相关 LOH 基因座的 LOH SNP 检测方法的原理验证。
