Identification and functional analysis of Rattus norvegicus Mammary carcinoma susceptibility 1b (Mcs1b) nominated variants.

Abstract

Rattus norvegicus (a.k.a. laboratory rat or Brown Rat) Mammary carcinoma susceptibility 1b (Mcs1b) is a concordant ortholog of a female breast cancer risk allele at human 5q11.2. Previously, Mcs1b was delimited to a 1.8 Mb interval of RNO2 and Map3k1 along with Mier3 were determined to be Mcs1b-nonminated genes. This conclusion was based on shared synteny with human 5q11.2 and differential gene expression between cancer susceptible and Mcs1b resistant mammary glands. In this study, targeted genome sequencing of cancer susceptible and Mcs1b resistance associated alleles was used to identify three Mcs1b-nominated quantitative trait nucleotides (QTNs) in noncoding DNA. In vitro approaches, luciferase activity and electromobility shift assays, were used to suggest these variants reside in potential gene regulatory elements. One of these variants, UL-A74-SNV-17, resulted in luciferase activities that were 2.6× higher for the susceptibility associated variant compared to the resistance associated variant. These results recapitulated Mcs1b nominated gene transcript level differences between Mcs1b genotypes in mammary epithelial cells (MECs), where Map3k1 and Mier3 were 1.5- to 2.0-fold higher for the susceptible genotype compared to the Mcs1b resistance-associated genotype. Evidence of a chromatin loop in Mcs1b that may position Mcs1b QTNs near distal genes was uncovered using chromosome confirmation capture (3C). Rat Mcs1b was also functionally characterized by determining that Mcs1b genotype had effects on the amount of luminal MECs in adult mammary glands. In conclusion, UL-A74-SNV-17 is a priority candidate Mcs1b QTN with a hypothesized mechanistic role in the differential regulation of Mcs1b nominated genes, Mier3 and Map3k1.