Functional Characterization of a Novel PBX1 De Novo Missense Variant Identified in a Pediatric Patient with CAKUT

Genetic variants in Pre-B cell Leukemia Factor 1 (PBX1) transcription factor (TF) have been associated with Congenital Anomalies of the Kidney and Urinary Tract (CAKUT). This study aims to functionally characterize a novel missense variant in a 4-year-old patient presenting with horseshoe kidney with preserved function, in the absence of a positive familial history.

Clinical exome sequencing was performed on a 4-year-old child, followed by Sanger sequencing and family segregation studies to validate the identified variant. Functional assays to study the protein expression, molecular interactions and localization were then performed.

Genetic analysis identified a novel de novo variant [c.712C>T, p.(Arg238Trp), NM_002585.3], mapping in the first nuclear localization signal (NLS) of PBX1. When introduced in HEK293T cells, PBX1^c.712C>T did not affect protein expression, which was comparable to the wild-type (WT) counterpart. Similar results were obtained when modeling a missense variant [c.863G>A; p.(Arg288Gln)], located in the second NLS of the protein, previously reported in the literature but never functionally characterized. As a TF, PBX1 may work in association with MEIS and PKNOX1/2 cofactors, but none of the two variants modified the interactions with its cofactor PKNOX1. However, both variants significantly affected the nuclear localization of PBX1, increasing its retention in the cytoplasm while limiting its availability in the nucleus.

In conclusion, we identified a novel de novo heterozygous missense variant in PBX1 that impairs nuclear localization of the protein, potentially limiting its role as a TF and possibly explaining the clinical phenotype of the patient.