A Mechanistic Evaluation of Naturally Occurring IGF1R 3’UTR Gene Variants Altering Disease Outcomes in Women

The insulin-like growth factor (IGF) axis, including its IGF1R receptor and IGF-1 ligand, is evolutionarily conserved and essential for normal organismal growth, development, and longevity. However, when dysregulated, this IGF growth axis can also drive disease and cancer development. In the human genome there are many naturally occurring IGF axis gene variants, especially within the IGF1R gene locus. A subset of these variants are single nucleotide polymorphisms (SNPs), and while none of these are known to be pathogenic, some (like rs2016347) are considered functional given their location within the IGF1R 3’ untranslated region (UTR), where they can potentially control tissue expression of IGF1R mRNA and protein. Prior population-based studies have shown that for reproductive age women diagnosed with either hypertensive disorders of pregnancy (HDP), endometriosis (ENDO), or polycystic ovary syndrome (PCOS), their risk for subsequently developing either cancer (including breast cancer), hypertension (HTN) or cardiovascular disease (CVD) is significantly reduced if they inherit the rs2016347 T allele. This allele has been shown in the GTEx database to be associated with significantly reduced IGF1R mRNA expression (relative to expression of its reference G allele) in the normal tissues and organs of healthy men and women. How inheritance of this seemingly protective rs2016347 T allele produces lower IGF1R mRNA levels at the molecular level remains unexplored and undefined. Therefore, in this study, our lab hypothesized that the IGF1R mRNA-regulating mechanism of rs2016347 was due to its alteration in the 3’UTR mRNA secondary structure, producing differential access to a transcript stability-regulating RNA binding protein and/or binding by a candidate microRNA (miR). To address this, we used bioinformatic analyses and in vitro cell culture studies to compare the rs2016347 T and G encoded variants with those of two other IGF1R 3’UTR SNPs in linkage disequilibrium (LD), rs2684788 and rs265498. These three SNPs in LD show similar GTEx IGF1R mRNA expression patterns in normal human tissues. Further consideration of rs265498 in determining GTEx IGF1R mRNA expression was discounted based on its RNAfold predicted SNP secondary structure comparisons, free energy calculations, and lack of nearby miR binding sites. Employing a small molecule inhibitor (BCI-137) of the miR-regulating Argonaut 2 protein in cell culture studies indicated the lack of any mechanistic involvement by the only pair of closely mapping candidate miRs (365A-3p and 455-5p) potentially capable of regulating IGF1R 3’UTR allele-specific gene expression. In contrast, by measuring mRNA and protein levels from luciferase reporting gene constructs bearing 3’UTR sequences from specific rs2016347 and rs2684788 allelic combinations transfected into HEK293 and SKBR3 human cell lines, produced clear evidence of IGF1R 3’UTR allele-specific gene expression patterns. While the T allele of rs2016347 produced significantly lower mRNA and protein reporter expression in the cell line models, this reduced expression was also totally dependent on C allele co-expression from rs2684788, indicating a requirement for cooperativity between these two LD SNPs in producing their population study observed protective outcome impacts. Future efforts are expected to re-evaluate earlier population-based studies and ask if accounting for both SNPs improves the disease outcome risk predictions previously determined using only the rs2016347 SNP. Likewise, cell culture mechanistic studies are planned that will attempt to detect a transcript destabilizing RNA binding protein (RBP) that only binds to the IGF1R 3’UTR when both the T allele of rs2016347 and the C allele of rs2684788 are expressed.