Poster 7
Presenter: David Samuelson
Wednesday, 3:00 – 5:00pm
David J. Samuelson, Jennifer Sanders, Xin Xu Center for Genetics & Molecular Medicine, Department of Biochemistry & Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky
Mammary carcinoma susceptibility-1b (Mcs1b) is a mammary-gland cell-autonomous ortholog of a human genome-wide association-study identified breast cancer risk locus at 5q11.2. Risk associated genetic variation is located in non-protein coding DNA for these concordant loci. Mesoderm induction early response 1, family member 3 (Mier3) is an Mcs1b candidate transcript exhibiting differential levels between cancer susceptible and resistant mammary glands. We hypothesize that Mcs1b is one or more noncoding genetic variants that function as gene regulatory elements. Targeted sequence capture and massively parallel sequencing of Mcs1b resistant and susceptible strain alleles was used to identify 70 single nucleotide variants (SNVs) and 2 insertion-deletions (INDELs) as potential quantitative trait nucleotides (QTNs). Sixty-seven SNVs and one INDEL were ruled out as independently acting susceptibility QTNs by genotyping congenic lines that were previously determined to not contain Mcs1b, but had either proximal or distal donor-strain alleles that bordered Mcs1b. To determine if SNVs in Mcs1b were regulatory elements we used luciferase reporter assays. Each SNV plus flanking sequences was independently cloned into pGL3-Promoter and transiently transfected into T47-D breast cancer cells. This epithelial-like cell line expresses endogenous MIER3. Immunohistochemical staining of mammary glands revealed that Mier3 protein was present in ductal epithelial cells. One SNV named A074-SNV-17 resulted in luciferase activities similar to differential mammary gland expression profiles between cancer susceptible and Mcs1b resistant genotypes. Oligonucleotides containing A074-SNV-17 sequence bound nuclear proteins. Mass spectrometry will be used to identify proteins that might bind to this SNV. In conclusion, A074-SNV-17 is a candidate Mcs1b QTN.