Functional Validation of a Novel PBX1 Missense Variant in a 46, XY Girl

Introduction: The pre-B-cell leukemia transcription factor encoded by PBX1 is expressed throughout human embryonic stages. Accumulating cases with differences of sex development (DSDs) have been reported harboring PBX1 variants, suggesting a yet elusive role of PBX1 in the gonadal differentiation and sexual development processes. Methods: We report a syndromic case of 46, XY DSD presenting severely undervirilized genitalia and gonadal dysgenesis with mixed ovarian and testicular differentiation, where whole-exome sequencing analysis identified a novel missense variant c.710G>C (p.R237T) in the highly conserved nuclear localization sequence in the three-amino acid loop extension domain of PBX1. Results: Compared with wild-type (WT) PBX1, PBX1 p.R237T reduced protein stability and hampered nuclear translocation of PBX1 in the inducible Flp-In TREx HEK293 cells. Induction with tetracycline significantly decreased cell proliferation in both Flp-In HEK293 PBX1 WT and p.R237T cells compared to untransfected HEK293 cells, while adhesive ability was not different. RNA sequencing identified differentially expressed genes in DSD-related genes, including MAP3K4, EMX2, KISS1R, and HOXA13 in cells expressing PBX1 p.R237T when compared to cells expressing WT PBX1. Conclusion: Altogether, our results demonstrated the deleterious functional consequences of the rare PBX1 p.R237T variant identified in a Taiwanese proband with 46, XY DSD.