Genetic linkage, genome mismatch scanning, and analysis of patients with alterations of chromosome 6 have indicated that a major locus for development of the anterior segment of the eye, IRID1, is located at 6p25. Abnormalities of this locus lead to glaucoma. FKHL7 (also called "FREAC3"), a member of the forkhead/winged-helix transcription-factor family, has also been mapped to 6p25. DNA sequencing of FKHL7 in five IRID1 families and 16 sporadic patients with anterior-segment defects revealed three mutations: a 10-bp deletion predicted to cause a frameshift and premature protein truncation prior to the FKHL7 forkhead DNA-binding domain, as well as two missense mutations of conserved amino acids within the FKHL7 forkhead domain. Mf1, the murine homologue of FKHL7, is expressed in the developing brain, skeletal system, and eye, consistent with FKHL7 having a role in ocular development. However, mutational screening and genetic-linkage analyses excluded FKHL7 from underlying the anterior-segment disorders in two IRID1 families with linkage to 6p25. Our findings demonstrate that, although mutations of FKHL7 result in anterior-segment defects and glaucoma in some patients, it is probable that at least one more locus involved in the regulation of eye development is also located at 6p25.

Mutations in the human FOXC1 transcription factor gene underlie Axenfeld-Rieger (AR) syndrome, a disorder characterized by anterior segment malformations in the eye and glaucoma. Through the use of an inducible FOXC1 protein, along with an intermediate protein synthesis blocker, we have determined direct targets of FOXC1 transcriptional regulation. FOXC1 regulates the expression of FOXO1A and binds to a conserved element in the FOXO1A promoter in vivo. The zebrafish foxO1a orthologs exhibit a robust expression pattern in the periocular mesenchyme. Furthermore, FOXO1A expression is reduced in cultured human trabecular meshwork (TM) cells and in the zebrafish developing eye when FOXC1 expression is knocked down by siRNAs and morpholino antisense oliognucleotides, respectively. We also demonstrate that reduced FOXC1 expression increases cell death in cultured TM cells in response to oxidative stress, and increases cell death in the developing zebrafish eye. These studies have uncovered a novel role for FOXC1 as an essential mediator of cellular homeostasis in the eye and indicate that a decreased resistance to oxidative stress may underlie AR-glaucoma pathogenesis. Given that FOXO1A influences cellular homeostasis when positively or negatively regulated; the dysregulation of FOXO1A activities in the eye through FOXC1 loss of function mutations and FOXC1 gene duplications provides an explanation into how seemingly similar human disorders can arise from both increases and decreases in FOXC1 gene dose.

Epstein-Barr virus (EBV) infection plays a major role in the pathogenesis of posttransplant lymphoproliferative disorder (PTLD). Quantitative oropharyngeal EBV shedding measured by a DNA-DNA dot blot assay and the genotype of isolates determined by a polymerase chain reaction assay were studied in 23 renal and 23 cardiac transplant recipients followed over the first posttransplant year. Five patients developed PTLD and two additional PTLD renal transplant recipients were studied from the time of diagnosis. Significantly higher levels of EBV were observed in primary versus reactivation infection (P < .04) when sequential courses of antilymphocyte globulins or > 4 g of methylprednisolone were used in the first 6 months after transplant and in patients with versus those without PTLD (P < .04), although the former group had a high incidence of primary infection. Patients with the highest EBV shedding had the poorest serologic responses. All PTLD patients shed EBV-1, which was also shed by patients without PTLD.

Mutations in the forkhead-like 7 (FKHL7) gene have been recently shown to cause juvenile glaucoma and anterior segment anomalies. We report on a three-generation family with Axenfeld-Rieger syndrome (ARS), harboring an alteration in the FKHL7 gene. Genetic linkage analyses excluded the ARS phenotype from chromosomes 4q25 and 13q14, the locations of the PITX2 and RIEG2 loci, respectively. Evidence of linkage was observed with markers at 6p25, near the FKHL7 gene. Direct sequencing of FKHL7 detected a C67T mutation that segregated with the ARS phenotype in this family, but was not detected in over 80 control chromosomes. This mutation is predicted to cause a nonsense mutation of the FKHL7 protein (Gln23Stop) upstream of the forkhead DNA-binding domain, and thus to generate a truncated FKHL7 protein product. This discovery broadly implicates FKHL7 in ocular, craniofacial, dental, and umbilical development.

Mutations in the FOXC1 transcription factor gene result in Axenfeld Rieger malformations, a disorder that affects the anterior segment of the eye, the teeth, and craniofacial structures. Individuals with this disorder possess an elevated risk for developing glaucoma. Previous work in our laboratory has indicated that FOXC1 transcriptional activity may be regulated by phosphorylation. We report here that FOXC1 is a short-lived protein (t1 ⁄ 2 <30 min), and serine 272 is a critical residue in maintaining proper stability of FOXC1. Furthermore, we have demonstrated that activation of the ERK1/2 mitogen-activated protein kinase through epidermal growth factor stimulation is required for maximal FOXC1 transcriptional activation and stability. Finally, we have demonstrated that FOXC1 is targeted to the ubiquitin 26 S proteasomal degradation pathway and that amino acid residues 367-553, which include the C-terminal transactivation domain of FOXC1, are essential for ubiquitin incorporation and proteolysis. These results indicate that FOXC1 protein levels and activity are tightly regulated by post-translational modifications.Protein phosphorylation provides a rapid means of altering the function of a protein in response to changes in the cellular environment. In the case of transcription factors, phosphorylation can alter the activity of these proteins through regulation of their nuclear localization (1, 2), modulation of their protein-protein (2, 3) and/or protein-DNA (4) interactions, and by controlling their stability (5). Therefore, the phosphorylation state of a transcription factor can dictate activity and act as a molecular switch from an inactive to an active form or vice versa.The Forkhead Box transcription factor FOXC1 is an integral component for the proper formation and function of structures derived from mesoderm and neural crest lineages (6 -10). In humans, mutations in the FOXC1 gene cause Axenfeld Rieger malformations, a disorder that is characterized by a spectrum of dysgeneses of the anterior segment of the eye (7,8). The most serious consequence of this disorder is a heightened propensity to develop glaucoma, with 50% of affected individuals developing this progressively blinding disease. In addition to the ocular findings, patients can present with a variable array of nonocular findings, including dental, craniofacial, umbilical, and cardiac anomalies. In mice, targeted deletion of both Foxc1 alleles results in neonatal lethality, hydrocephalus, ocular and skeletal abnormalities (6, 10 -12), establishing Foxc1 as an essential developmental transcription factor.Previous work in our laboratory has demonstrated that the FOXC1 transcription factor is a phosphoprotein and that amino acid residues 215-366 contribute to a phosphorylation-dependent mobility shift in the FOXC1 protein as detected by SDS-PAGE (13). Removal of this region prevents this mobility shift and results in a transcriptionally hyperactive FOXC1 molecule. We sought to identify the residues that are phosphorylated in FOXC1 and the kinases ...