Molecular Characterization of Novel FOXN1 Mutations Causal to Thymic Aplasias in Human Patients

The Forkhead Box N1 (FOXN1) transcription factor plays a crucial role in thymic epithelial cell development. Humans and mice harboring FOXN1 mutations have a profound T-cell deficiency caused by their thymic aplasia. They also present with alopecia and nail dystrophy. Recently, two patients were identified with T-cell immunodeficiency. Both patients have normal hair and nailbed development. Genetic workup revealed that each patient carried distinct compound heterozygous mutations in FOXN1 not previously reported. Molecular characterization of these FOXN1 mutations will provide new insight into how this transcription factor functions in thymus development. To characterize these mutations, CRISPR/Cas9 technologies were used to create similar compound heterozygous mutations in mouse models. The mice are currently being intercrossed to determine the impact of these novel FOXN1 mutations on thymus development. To determine how these mutations impact FOXN1 function, we undertook transcriptional reporter assays. Preliminary results suggest only one of these mutations led to loss of transcriptional activity. Western blot analysis indicated that this mutation led to a truncation of the protein. Further experiments including co-immunoprecipitation assays, transcriptome analyses, and functional studies will reveal how these compound heterozygous mutations impact the functions of FOXN1. Findings from this study may lay the foundation for novel therapeutic strategies at restoring thymopoiesis in a number of distinct clinical settings. These can include patients undergoing radiation treatment, chemotherapy, and in any other conditions that can lead to a thymic aplasia.