Validation of prostate cancer risk variants by CRISPR/Cas9 mediated genome editing

Abstract

GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the MSMB promoter, associates with MSMB encoded β-microseminoprotein prostate secretion levels, and is associated with mRNA expression changes in MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in LD 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 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 summaryIn pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in 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 tissue 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.

SNPMap—An integrated visual SNP interpretation tool

“…As shown in Table 3, LitVar keywords are more concise and precise, while SNPMap's keywords are often expanded and supplemented based on the most important keywords. While some keywords related to disease, chemicals, and variants are displayed on the sidebar of the web page, the location made the keywords less intuitive and precise, while missing out on some important concepts (e.g., prostate-specific antigen (PSA) for rs10993994, since it is previously reported association of the SNP (Wiklund et al, 2009;Wang et al, 2021)). The graph on SNPMap provides additional information that is not represented on LitVar that includes keyword connections that have strong connections among concepts of "prostate cancer", and "MSMB" (the gene where the SNP is located), "prostate-related antigen" (closely related to prostate cancer).…”