Haploinsufficient phenotypes promote selection of PTEN and ARID1A deficient clones in human colon.. Haploinsufficient phenotypes promote selection of PTEN and ARID1A deficient clones in human colon.

Normal human tissues are known to be composed of a pactchwork of mutant clones but with limited implications on cancer risk. PTEN and ARID1A deficient clones were detected in the human colon using immunohistrochemistry and were found to be the product of monoallelic loess. They were associated with a bias in clone dynamics which resulted in positive selection in the tissue. Heterozygous mutations were introduced in intestinal organoids which were subjected to bulk RNA-seq to describe the molecular mechanisms behind their positive selection. Although PTEN het organoids were associated with an advantageous phenotype in organoids, which was recapitulated in the transcriptomic analysis with increase proliferation and upregulation of developmental/regenerative processes and metabolic reprogramming, the ARID1A het organoids showed reduced growth which was seen as upregulation of apoptosis and downregulation of DNA replication. The discrepancy between the tissue and organoid phenotype for ARID1A was later explained due to the impact on stromal cells which are found in the tissue and are mediating the selective advantage. Importantly these molecular alterations and behaviours arise due to haploinsufficiency of these tumour suppressors in a pre-neoplastic context. Overall design: We subjected the generated heterozygous organoids to bulk RNA-seq to study the molecular effects of the monoallelic gene loss. PTEN het, ARID1A het, WT and PTEN KO- 4 replicates each that were different isogenic clones

已知正常人体组织由携带突变的克隆构成的镶嵌体，但其对癌症风险的影响较为有限。研究人员通过免疫组化（immunohistochemistry）技术在人类结肠组织中检测到PTEN与ARID1A缺陷型克隆，证实此类克隆源于单等位基因缺失。该类克隆表现出克隆动态偏倚特征，进而在组织中受到正向选择。
为解析其正向选择背后的分子机制，研究人员向肠道类器官引入杂合突变，并通过批量RNA测序（bulk RNA-seq）开展相关分析。尽管PTEN杂合（PTEN het）类器官呈现出优势表型，转录组分析显示其增殖能力增强、发育/再生相关过程基因上调且伴随代谢重编程，该结果与类器官层面的表型观察一致；而ARID1A杂合（ARID1A het）类器官则表现出生长减缓，转录组特征为细胞凋亡相关基因上调、DNA复制相关基因下调。
后续研究表明，ARID1A杂合类器官与组织层面的表型差异，源于组织中基质细胞所介导的选择优势对该类克隆的调控作用。
值得注意的是，上述分子改变与表型行为，均源于癌前环境下这些抑癌基因的单倍体剂量不足（haploinsufficiency）效应。
实验整体设计：本研究将构建获得的杂合类器官进行批量RNA测序，以探究单等位基因缺失的分子效应。实验分组涵盖PTEN杂合组、ARID1A杂合组、野生型（WT）组与PTEN基因敲除（PTEN KO）组，每组均设置4个生物学重复，且每个重复均来自不同的同基因克隆（isogenic clones）。