Background DICER1 syndrome is an autosomal dominant inherited syndrome predisposing to various benign and malignant tumors, mainly occurring in children and young adults, requiring broad surveillance starting at birth with repeated irradiating imaging exams and sedations for young patients. It is caused by monoallelic germline pathogenic variants in the DICER1 gene. More than 90% of tumors bear an additional somatic DICER1 missense hotspot mutation, as a second hit, involving 1 of 6 codons clustered in exons 24 and 25. We designed and in vitro validated a drop-off droplet digital PCR (ddPCR) system to scan all DICER1 hotspot codons, allowing for a liquid biopsy test, an alternative to sedation and radiation exposure. Methods Three drop-off ddPCR assays were designed, with 2 TaqMan probes per assay, 1 complementary to the wild-type sequence of the region containing hotspots and another 1 used as a reference. Eight tumor-derived DNAs and 5 synthetic oligonucleotides bearing DICER1 hotspot mutations were tested. Results All tested mutations were detected, with a limit of detection ranging from 0.07% to 0.31% for codons p. E1705, p. D1709, and p. D1713 in exon 24 and from 0.06% to 0.15% for codons p. G1809, p. D1810, and p. E1813 in exon 25. Conclusions The high sensitivity of this method is compatible with its use for plasma circulating tumor DNA (ctDNA) analysis for early tumor detection in DICER1 syndrome patients. It may reduce the need for radiation exposure and sedation in surveillance protocols and may also improve patient prognosis. Clinical trials are needed to evaluate ctDNA analysis in these patients.

中文翻译：

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Drop-off Droplet Digital PCR 高灵敏度检测 DICER1 肿瘤热点突变的方法

背景 DICER1 综合征是一种常染色体显性遗传综合征，易患各种良性和恶性肿瘤，主要发生在儿童和年轻人中，需要从出生开始进行广泛监测，对年轻患者进行反复照射成像检查和镇静剂。它是由 DICER1 基因中的单等位基因种系致病变异引起的。超过 90% 的肿瘤具有额外的体细胞 DICER1 错义热点突变，作为第二次打击，涉及聚集在外显子 24 和 25 中的 6 个密码子中的 1 个。我们设计并在体外验证了一个滴滴式数字 PCR (ddPCR) 系统扫描所有 DICER1 热点密码子，允许进行液体活检测试，这是镇静和辐射​​暴露的替代方法。方法 设计了三个 drop-off ddPCR 检测，每个检测使用 2 个 TaqMan 探针，1个与包含热点的区域的野生型序列互补，另外1个用作参考。测试了八种来自肿瘤的 DNA 和 5 种带有 DICER1 热点突变的合成寡核苷酸。结果所有检测到的突变均被检测到，密码子p的检测限为0.07%至0.31%。E1705，页。D1709 和 p。外显子 24 中的 D1713 和密码子 p 的 0.06% 至 0.15%。G1809，第 D1810 和 p。E1813 在外显子 25 中。结论该方法的高灵敏度与其用于 DICER1 综合征患者早期肿瘤检测的血浆循环肿瘤 DNA (ctDNA) 分析相兼容。它可以减少监测方案中对辐射照射和镇静的需求，还可以改善患者预后。需要临床试验来评估这些患者的 ctDNA 分析。