Functional Assessment of a New PBX1 Variant in a 46,XY Fetus with Severe Syndromic Difference of Sexual Development through CRISPR-Cas9 Gene Editing

Genes (Basel). 2023 Jan 20;14(2):273. doi: 10.3390/genes14020273.

Abstract

Sexual development is a complex process relying on numerous genes. Disruptions in some of these genes are known to cause differences of sexual development (DSDs). Advances in genome sequencing allowed the discovery of new genes implicated in sexual development, such as PBX1. We present here a fetus with a new PBX1 NM_002585.3: c.320G>A,p.(Arg107Gln) variant, presenting with severe DSD along with renal and lung malformations. Using CRISPR-Cas9 gene editing on HEK293T cells, we generated a KD cell line for PBX1. The KD cell line showed reduced proliferation and adhesion properties compared with HEK293T cells. HEK293T and KD cells were then transfected plasmids coding either PBX1 WT or PBX1-320G>A (mutant). WT or mutant PBX1 overexpression rescued cell proliferation in both cell lines. RNA-seq analyses showed less than 30 differentially expressed genes, in ectopic mutant-PBX1-expressing cells compared with WT-PBX1. Among them, U2AF1, encoding a splicing factor subunit, is an interesting candidate. Overall, mutant PBX1 seems to have modest effects compared with WT PBX1 in our model. However, the recurrence of PBX1 Arg107 substitution in patients with closely related phenotypes calls for its impact in human diseases. Further functional studies are needed to explore its effects on cellular metabolism.

Keywords: CRISPR-Cas9; DSD; RNA-seq; genome editing; sexual development.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Fetus
  • Gene Editing*
  • HEK293 Cells
  • Humans
  • Pre-B-Cell Leukemia Transcription Factor 1 / genetics
  • Sexual Development

Substances

  • PBX1 protein, human
  • Pre-B-Cell Leukemia Transcription Factor 1

Grants and funding

This study received financial support from AVIESAN Plan Cancer, Région Bretagne; Agence Nationale de Recherche, University of Rennes 1 and Ministère de la Recherche et de l’Enseignement Supérieur (MESRI, PhD Scholarship).