Department of Genetics and Genomic Sciences and Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, United States.
Department of Genetics and Genomic Sciences and Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, United States; Program in Cancer Biology and Genetics and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, United States; Graduate School of Biomedical Sciences, Weill Cornell Medical College, New York, NY, United States.
Gene. 2021 Feb 5;768:145265. doi: 10.1016/j.gene.2020.145265. Epub 2020 Oct 26.
GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the β-microseminoprotein (MSMB) promoter region, mediates MSMB prostate secretion levels, and is linked to mRNA expression changes in both MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in positive linkage disequilibrium with rs10993994 and associated with MSMB expression independent of rs10993994. Here, we generate a series of clones with single alleles removed by double guide RNA (gRNA) mediated CRISPR/Cas9 deletions, through which we demonstrate that each of these SNPs independently and greatly alters MSMB expression in an allele-specific manner. We further show that these SNPs have no substantial effect on the expression of NCOA4. These data demonstrate that a single SNP can have a large effect on gene expression and illustrate the importance of functional validation studies to deconvolute observed correlations. The method we have developed is generally applicable to test any SNP for which a relevant heterozygous cell line is available. AUTHOR SUMMARY: In pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in the human genome as genetic markers to perform large scale genome wide association studies (GWAS) and have so far identified more than a hundred prostate cancer risk variants. Such variants provide an unbiased and systematic new venue to study the disease mechanism, and the next big challenge is to translate these genetic associations to the causal role of altered gene function in oncogenesis. The majority of these variants are waiting to be studied and lots of them may act in oncogenesis through gene expression regulation. To prove the concept, we took rs10993994 and its linked rs7098889 as an example and engineered single cell clones by allelic-specific CRISPR/Cas9 deletion to separate the effect of each allele. We observed that a single nucleotide difference would lead to surprisingly high level of MSMB gene expression change in a gene specific and cell-type specific manner. Our study strongly supports the notion that differential level of gene expression caused by risk variants and their associated genetic locus play a major role in oncogenesis and also highlights the importance of studying the function of MSMB encoded β-MSP in prostate cancer pathogenesis.
全基因组关联研究（GWAS）已经确定了许多与前列腺癌风险相关的单核苷酸多态性（SNP）。其中一个 SNP 是 rs10993994。它位于β-微精蛋白（MSMB）启动子区域，介导 MSMB 前列腺分泌水平，并与 MSMB 和相邻基因 NCOA4 的 mRNA 表达变化相关。此外，我们之前的工作表明，第二个 SNP rs7098889 与 rs10993994 呈正连锁不平衡，并与 rs10993994 无关，与 MSMB 表达相关。在这里，我们通过双向导 RNA（gRNA）介导的 CRISPR/Cas9 缺失产生了一系列缺失单个等位基因的克隆，通过这些克隆，我们证明这些 SNP 中的每一个都以等位基因特异性的方式独立且极大地改变了 MSMB 的表达。我们进一步表明，这些 SNP 对 NCOA4 的表达没有实质性影响。这些数据表明，单个 SNP 可以对基因表达产生很大的影响，并说明了功能验证研究对于剖析观察到的相关性的重要性。我们开发的方法通常适用于测试任何具有相关杂合细胞系的 SNP。
在探索前列腺癌发病机制的潜在生物学机制时，科学家们利用人类基因组中存在的常见单核苷酸多态性（SNP）作为遗传标记，进行了大规模全基因组关联研究（GWAS），迄今为止已确定了 100 多种前列腺癌风险变体。这些变体为研究疾病机制提供了一个无偏见和系统的新途径，下一个大挑战是将这些遗传关联转化为改变基因功能在肿瘤发生中的因果作用。大多数这些变体仍有待研究，其中许多可能通过基因表达调控在肿瘤发生中起作用。为了证明这一概念，我们以 rs10993994 及其连锁的 rs7098889 为例，通过等位基因特异性 CRISPR/Cas9 缺失工程构建了单细胞克隆，以分离每个等位基因的作用。我们观察到，单个核苷酸差异会导致 MSMB 基因表达水平以基因特异性和细胞类型特异性的方式发生惊人的变化。我们的研究强烈支持这样一种观点，即风险变体及其相关遗传位点引起的基因表达水平的差异在肿瘤发生中起着重要作用，同时也突出了研究 MSMB 编码的β-MSP 在前列腺癌发病机制中的功能的重要性。
