(P18-056-23) Genetic Variants in MFGE8 Have Minimal Effects on the Mammary Epithelial Cell Transcriptome

Objectives: Breastfeeding provides numerous benefits to both mothers and infants; however, ~60% of mothers stop breastfeeding earlier than planned due to concerns over low milk supply (LMS). Genetic variation in MFGE8 is associated with LMS. MFGE8 is a secreted glycoprotein that plays a role in cell adhesion and assists in the clearance of apoptotic cells. However, consequences of genetic variants in MFGE8 on mammary gland function have not been explored. Here we explored effects of two common MFGE8 variants (L132V and L76M) on the mammary epithelial cell (MEC) transcriptome to determine how these variants affect MEC function.

Methods: Site directed mutagenesis was used to generate plasmid DNA containing L132V (#rs2271714) and L76M (#rs1878326) and mutations were confirmed by DNA sequencing. Cultured MECs (HC11 cells) were transfected to express wild-type (WT) MFGE8 or each variant, and expression was confirmed by immunoblotting. RNA was isolated (n=4 samples/group) and RNAseq was used to identify differentially expressed genes. Enrichment of signaling pathway analysis was performed based on the Gene Ontology (GO) database.

Results: MFGE8 overexpression resulted in differential expression of 39 genes. The GO Biological Processes significantly affected ( >5 genes) included metabolic processes, macromolecule and protein modification, catabolic process, and lipid metabolism. Key genes most highly and significantly upregulated were BIVM, CUL9, GPR137, EIFE4E, ADH7, and TIRC6C, while PIKC32B, DDX58, OAS3, DIS3I, SLC25A22, and SAMD9I were downregulated.  The only genes differentially expressed by the MFGE8 variants were DPY19L3, PFKP and SAMD91 (L132V) and DPY19L3 (L76M).

Conclusions: These results indicate MFGE8 expression alters protein, lipid and carbohydrate processing and metabolism in MECs; however, neither MFGE8 variant had substantial effects on the transcriptome. This excludes the possibility that genetic variants in MFGE8 impair MEC function directly and indicates studies to understand consequences on extracellular functions and cell-cell interactions are required to understand their association with low milk supply.

Funding Sources: UMass Lowell Internal Seed Fund