Defining Genotype to Phenotype Causality in Patients with Diverse FOXN1 Variants

Background Forkhead box N1 (FOXN1) is the master transcriptional regulator of thymic epithelial cells (TECs), quintessential for T cell development. Well-defined biallelic FOXN1 loss-of-function variants lead to congenital athymia. However, most patients who present with low T cell numbers due to FOXN1 mutations have single allelic variants, the majority of which are of unknown significance. For some individuals, their T cell numbers can improve over a prolonged period, often months to years. The mechanistic basis by which single-allele FOXN1 variants can disrupt thymopoiesis remains poorly understood. To address this knowledge gap, we developed a multi-platform strategy to define how specific human FOXN1 mutations cause T cell lymphopenia due to TEC dysfunctions. Methods Human FOXN1 variants were screened for transcriptional activity with reporter assays, cytoplasmic versus nuclear distributions, and the potential for aggregation or co-association. Allelic expression patterns are currently being assessed with mouse models developed to genocopy selected human FOXN1 mutations. Results Transcriptional reporter assays effectively categorize human FOXN1 variants into complete, partial, and gain-of-function mutations. Both partial loss- and gain-of-function can reduce thymic T cell output. A subset of the human FOXN1 variants function as dominant negatives, antagonizing the function of the wild-type allele. However, this accounts for the effects of a subset of variants, indicating that other mechanisms are involved. Current experiments are addressing whether particular human FOXN1 variants express more stable mRNA transcripts that compete with the normal transcript and/or whether this gene is governed by monoallelic expression. Conclusions Human FOXN1 mutations can be effectively categorized into loss- and gain-of-function, which explains the clinical phenotype of low T cell numbers due to thymic hypoplasia. However, other allelic variants can function as dominant negative or may undergo a monoallelic expression, also leading to a T cell lymphopenia. Such a combinatorial approach will lead to better classifications, enabling more informed clinical interventions.