Characterization of a novel FOXC1 mutation, P297S, identified in two individuals with anterior segment dysgenesis.

To the Editor: FOXC1 is a member of the forkhead transcription factor gene family as defined by the presence of the DNA binding forkhead domain. Mutations in FOXC1 lead to Axenfeld–Rieger syndrome, a disease characterized by ocular phenotypes and increased glaucoma risk (1). Mutations resulting in reduced FOXC1 function and duplications of FOXC1 both cause disease, indicating that precise regulation of FOXC1 transcriptional targets is required for normal eye development and maintenance (1). There have been 22 missense mutations at 18 different amino acids, all within the forkhead domain of FOXC1, in patients with ocular abnormalities identified to date (Fig. 1a). Here, the first FOXC1 missense mutation, P297S, that occurs outside of the forkhead domain is reported and functionally examined. The 889C>T transition, resulting in P297S, was identified in two unrelated individuals with anterior segment dysgenesis (Fig. 1b,c), but not in 50 control subjects, through sequencing of FOXC1. P297 is located within the inhibitory domain of FOXC1, a phosphorylated region that is unstructured. The P297S mutation did not affect protein localization to the nucleus or DNA binding, functions attributed to the forkhead domain (Fig. 1d,e). In addition, FOXC1 P297S expression levels and molecular weight were not altered compared to wild-type (Fig. 1f), indicating that differences in post-translational modifications between FOXC1 P297S and wild-type are unlikely. The inhibitory domain is involved in the regulation of protein degradation (2), and FOXC1 P297S was found to have a half-life that is 45% longer than wild-type (Fig. 1g). Phosphorylation of S272 prevents degradation while a C-terminal degron is required for efficient degradation (2). P297S may potentially alter or interfere with these or other unidentified degradation or stabilization signal(s). The inhibitory domain also functions as transactivation inhibitor (1). Correct FOXC1 function requires transactivation ability in the range of 78–150% of wild-type activation (3). The transactivation ability of P297S was consistently less than 75% of wild-type on three different reporter constructs that FOXC1 is known to activate (Fig. 1h) (3–5). While the decrease in transactivation ability of FOXC1 P297S was not dramatic, it is likely biologically significant since FOXC1 is known to have very stringent upper and lower thresholds for normal activity (3). P297S is the first missense mutation identified outside of the forkhead domain in FOXC1. It is a recurrent mutation as it was present in two unrelated individuals with anterior segment dysgenesis. FOXC1 is under tight regulatory control, thus any disturbances to this regulation are likely pathogenic. P297 is located within the inhibitory domain of FOXC1, a region known to be important for regulating transactivation activity and protein stability, and a reduction in transactivation ability and extended half-life for FOXC1 P297S were demonstrated. It is likely that P297S alters FOXC1 interaction with other yet unidentified factors involved in transactivation and degradation. Both of these alterations alone may be disease causing and their effects may be tissue dependent and timing of expression dependent. Missense mutations outside of the forkhead domain have also been identified in the forkhead genes FOXA2, FOXE1, FOXI1, FOXL2 and FOXP2. The molecular consequences of missense mutations found outside of the forkhead domain have been briefly examined in FOXA2, FOXI1 and FOXL2 (6–9). The nuclear localization of mutant FOXI1 and FOXL2 was shown to be normal, and the transactivation ability of FOXA2, FOXI1 and FOXL2 mutants was altered compared to wildtype, with the exception of one FOXL2 mutant,