Hereditary Gastrointestinal Cancer Genetic Diagnosis Laboratory, Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong.
Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong.
Gut. 2017 Sep;66(9):1645-1656. doi: 10.1136/gutjnl-2016-311849. Epub 2016 Jun 21.
Serrated polyps (hyperplastic polyps, sessile or traditional serrated adenomas), which can arise in a sporadic or polyposis setting, predispose to colorectal cancer (CRC), especially those with microsatellite instability (MSI) due to MLH1 promoter methylation (MLH1). We investigate genetic alterations in the serrated polyposis pathway.
We used a combination of exome sequencing and target gene Sanger sequencing to study serrated polyposis families, sporadic serrated polyps and CRCs, with validation by analysis of The Cancer Genome Atlas (TCGA) cohort, followed by organoid-based functional studies.
In one out of four serrated polyposis families, we identified a germline RNF43 mutation that displayed autosomal dominant cosegregation with the serrated polyposis phenotype, along with second-hit inactivation through loss of heterozygosity or somatic mutations in all serrated polyps (16), adenomas (5) and cancer (1) examined, as well as coincidental BRAF mutation in 62.5% of the serrated polyps. Concurrently, somatic RNF43 mutations were identified in 34% of sporadic sessile/traditional serrated adenomas, but 0% of hyperplastic polyps (p=0.013). Lastly, in MSI CRCs, we found significantly more frequent RNF43 mutations in the MLH1 (85%) versus MLH1 (33.3%) group (p<0.001). These findings were validated in the TCGA MSI CRCs (p=0.005), which further delineated a significant differential involvement of three Wnt pathway genes between these two groups (RNF43 in MLH1; APC and CTNNB1 in MLH1); and identified significant co-occurrence of BRAF and RNF43 mutations in the MSI (p<0.001), microsatellite stable (MSS) (p=0.002) and MLH1 MSI CRCs (p=0.042). Functionally, organoid culture of serrated adenoma or mouse colon with CRISPR-induced RNF43 mutations had reduced dependency on R-spondin1.
These results illustrate the importance of RNF43, along with BRAF mutation in the serrated neoplasia pathway (both the sporadic and familial forms), inform genetic diagnosis protocol and raise therapeutic opportunities through Wnt inhibition in different stages of evolution of serrated polyps.
锯齿状息肉（增生性息肉、无蒂或传统锯齿状腺瘤）可在散发或息肉病环境中发生，易发生结直肠癌（CRC），尤其是那些由于 MLH1 启动子甲基化（MLH1）而具有微卫星不稳定性（MSI）的息肉。我们研究锯齿状息肉形成途径中的遗传改变。
我们使用外显子组测序和靶向基因 Sanger 测序的组合来研究锯齿状息肉家族、散发锯齿状息肉和 CRC，通过分析癌症基因组图谱（TCGA）队列进行验证，然后进行基于类器官的功能研究。
在四个锯齿状息肉家族中的一个家族中，我们发现了一个种系 RNF43 突变，该突变与锯齿状息肉表型呈常染色体显性共分离，并通过杂合性丢失或所有锯齿状息肉（16 个）、腺瘤（5 个）和癌症（1 个）的体细胞突变失活，以及 62.5%的锯齿状息肉中同时发生 BRAF 突变。同时，在 34%的散发无蒂/传统锯齿状腺瘤中发现了体细胞 RNF43 突变，但在增生性息肉中为 0%（p=0.013）。最后，在 MSI CRC 中，我们发现 MLH1（85%）组中 RNF43 突变的频率明显高于 MLH1（33.3%）组（p<0.001）。这些发现在 TCGA MSI CRC 中得到了验证（p=0.005），进一步阐明了这两组之间三个 Wnt 通路基因的显著差异参与（MLH1 中的 RNF43；APC 和 MLH1 中的 CTNNB1）；并确定了 BRAF 和 RNF43 突变在 MSI（p<0.001）、微卫星稳定（MSS）（p=0.002）和 MLH1 MSI CRC 中的显著共同发生（p=0.042）。功能上，CRISPR 诱导的 RNF43 突变的锯齿状腺瘤或小鼠结肠类器官培养显示对 R- 反应蛋白 1 的依赖性降低。
这些结果说明了 RNF43 以及 BRAF 突变在锯齿状肿瘤发生途径中的重要性（包括散发和家族形式），为遗传诊断方案提供了信息，并通过在锯齿状息肉不同进化阶段抑制 Wnt 提供了治疗机会。
