Postdoctoral fellow German Cancer Research Center (DKFZ) Heidelberg, Germany
Introduction: Several genome-wide association studies (GWAS) have been conducted to identify germline variants predisposing to multiple myeloma (MM). Up to date, 24 loci were found to be associated with MM risk, but very little information is available about their functional role. GWAS design takes advantage of the linkage disequilibrium (LD) structure of the human genome, thus the main GWAS findings are single-nucleotide polymorphisms (SNPs) that show the strongest association with MM risk (measured as the lowest p-values), but they are not necessarily the functionally causal variants. The functional characterization of the causal risk variants would lead to a better understanding of disease development. For these reasons we aimed at exploring in vitro the function of the GWAS-identified rs3747481 SNP through CRISPR-Cass9 knock-in technology in the U266B1 MM cell line.
Methods: To maximize the probability to capture the casual variant, we focused on chr16 q23.1, the locus with the smallest number of SNPs in high LD (r2>0.8). A cell pool of U266B1 containing the edited locus of interest was generated by the company Synthego (https://www.synthego.com/) with CRISPR-Cas9 knock-in technology. The isogenic cell colonies were then generated using a CloneSelect Single-Cell Printer. Successfully growing colonies with the three possible genotypes at rs3747481 were functionally tested. In particular, we measured: differences in proliferation, migration and gene expression of the nearby gene RNF40. The last test was also performed in 25 MM bone marrow samples with known genotypes at rs3747481. Kruskal-Wallis and T test were used to analyse the statistical significance.
Results: rs3747481 was of particular functional interest, due to the following reasons: it is a missense variant (protein change: P359L), has a high CADD PHRED score (22.1) and, according to GTEx portal, it is associated with the expression level of the RNF40 gene. We successfully isolated two T/T colonies and one C/T colony for functional testing (T is the MM risk associated allele). A 2.3-fold decrease in proliferation was observed in T/T colonies and 2.9 for colonies with C/T genotype compared to the C/C wild type (p=0.02). The migration assay showed a significant increase in cell migration of 3-fold (p=0.002) for the cells carrying the T allele. A significant increase of the expression of RNF40 was also observed when comparing the 3 genotypes in the cell lines, with the highest expression in T/T cells (p=0.001). A similar difference was observed in the BM patients as well, although not significant.
Conclusions: We successfully edited the genotypes of interest and observed a decrease in proliferation and an increase in migration in colonies with the MM risk allele, as well as a significant increase in the expression of RNF40. These findings confirmed the functional role of rs3747481. However, further experiments will be needed to better understand the biological mechanism of action of the polymorphism.
