Forkhead Box C1 Gene Research Articles

Genetic analysis of patients with primary congenital glaucoma.

To determine the common gene mutation in patients with primary congenital glaucoma (PCG) in the Southeast region of Turkey via genetic analysis and to evaluate whether there were other gene mutations in these patients. A total of 25 patients with PCG were included in this study. We performed sequence analysis including all exons of cytochrome p450 1B1 (CYP1B1), myocilin (MYOC), forkhead box C1 (FOXC1), and paired-like homeodomain 2 (PITX2) genes of the obtained samples. Further, we analyzed the results using the Nextgen analysis program. The CYP1B1 gene mutation was detected in 20 (80%) of 25 patients, and FOXC1 gene mutation was detected in one (4%) patient. The mutation site of nine (45%) of the 20 CYP1B1 genes was found in the second exon. The pathogenic variant (p.Gly61Glu) was observed in 12 (60%) patients (in the first and second exons); the mutation type of six (50%) of these patients was homozygous. The mutation site of one patient with FOXC1 gene mutation was found to be in the first exon; its pathogenic variant was p.Met400lle. The mutation type in this gene was observed to be heterozygous. Lastly, there were no mutations in the MYOC, FOXC1, and PITX2 genes in combination with the CYP1B1 gene mutation. The most common cause of PCG in our region is the CYP1B1 gene mutation, and the most frequent pathogenic variant is c.182G > A (p.Gly61Glu). We also determined that the CYP1B1 gene mutation was alone and did not occur with other gene mutations (MYOC, FOXC1, and PITX2).

MON-257 Axenfeld Rieger Syndrome: An Uncommon Cause of Growth Hormone Deficiency

Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder presenting with abnormal eye development, which leads to glaucoma related blindness in 50% of individuals. Associated mutations affect the transcription factors pituitary homeobox 2 gene (PITX2) and forkhead box C1 gene (FOXC1). Three types of ARS have been described. PITX2 mutation causes ARS type I, which is associated to systemic malformations, including dental hypoplasia, redundant periumbilical skin, and growth hormone deficiency (GHD).This is the case of a 28-year-old male diagnosed with GHD during childhood. He was referred to a pediatric-endocrinologist at age 10 due to short stature. Evaluation showed a height-for-age curve below the 10th percentile. Physical examination with prominent forehead, decrease visual acuity, maxillary hypodontia, umbilical hernia, and delayed sexual maturity. Testing with reduced IGF-1 and delayed bone age. Clonidine GH stimulation confirmed the diagnosis of GDH. He was treated with somatropin, until linear growth decreased to ½ inch per year at age 16. GHD etiology was never established. At age 26, he developed progressive decrease in visual acuity. Ophthalmology evaluation disclosed polycoria, megalocornea and increase intraocular pressure, suggestive of ARS. Patient was referred to our endocrinology clinics for follow up of previous diagnosis of GHD. Based on clinical findings and history, sequence analysis and deletion/duplication testing of FOXC1, PAX6 and PITx2 were performed, with results positive for pathogenic variant PITX2, Exon 5, c.363_364delinsAA. Assessment of pituitary hormone axis was normal, and no persistent GHD found. No family members exhibited clinical signs of ARS.Axenfeld-Rieger syndrome is a rare genetic disease. PITX homeodomain transcription factors are critical for the development of the anterior segment of the eye and pituitary. Most mutations in PITX2 affect DNA binding and transactivation that leads to defects in cell proliferation and differentiation of the Rathke’s pouch. As a result, GHD may ensue. ARS patients are usually diagnosed during childhood after the development of vision abnormalities. The diagnosis remains primarily clinical upon identification of ocular abnormalities in the iris and cornea, and increased intraocular pressure. Systemic changes are rare findings in ARS, but may include face and tooth abnormalities and isolated growth hormone deficiency. Genetic diagnosis is based on identification of mutations. An adequate management of ARS requires a multidisciplinary approach. Although ophthalmologists usually diagnose this condition, some patients initially present with isolated growth impairment. This may lead to a delay in ophthalmologic evaluation and management. Thus, in patients with GHD of unknown etiology, it is important to have a high index of suspicion of ARS in order to decrease morbidity from vision loss.

Forkhead box C1 gene variant causing glaucoma and small vessel angiopathy can mimic multiple sclerosis

A 34–year old Caucasian female was initially diagnosed with multiple small-vessel strokes at age 20 years which were etiologically classified as secondary to anti-phospholipid antibody syndrome (APS) although she had no history or laboratory data to suggest APS. Based on her MRI of brain findings, one of her neurologists was concerned she could have multiple sclerosis (MS) and hence the patient was referred to our clinic for further evaluation. The patient's MRI of brain showed confluent lesions in the periventricular and juxta-cortical lesions that fulfil 2017 McDonald criteria for dissemination in space. She had no symptoms other than occasional, mild headaches and had no findings to suggest clinically isolated syndrome (CIS) or MS; additionally, her cerebrospinal fluid analysis was unremarkable. Past history showed that she had undergone surgery for glaucoma, and subsequently developed bilateral sensorineural hearing loss in the third decade that was diagnosed as Meniere's disease. Her family history revealed that her son had dysmorphic facies and was small for age. He had a bifid uvula, bilaterally duplicated thumbs and scoliosis. Additionally, he had hypertelorism, a wide forehead and flattening of mid-face. Due to his complex medical presentation, whole exome sequencing (WES) was performed that revealed a maternally inherited heterozygous pathogenic frameshift in the FOXC1 gene. Genotyping of the mother showed the FOXC1 gene variant and adds to the growing list of differential diagnoses that may mimic MS in the context of radiological changes involving cerebral small vessels. This is the first report of a FOXC1 gene variant presenting with radiological features that can erroneously be interpreted as being consistent with MS.

FOXC1 is Over‐expressed and is More Stable in Triple Negative/Basal‐Like Breast Cancer

Rapidly accumulating evidence implicates forkhead box C1 (FOXC1) in Triple Negative Breast Cancer (TNBC), particularly in Basal‐Like Breast cancer (BLBC). Recently additional studies have demonstrating that FOXC1 is also a major player in hepatocellular carcinoma (HCC), endometrial cancer, Hodgkin's lymphoma (HL), non‐Hodgkin's lymphoma (NHL), and others. The FOXC1 gene encodes a transcription factor that is crucial to mesodermal, neural crest, and ocular development. Loss of function mutations in FOXC1 have been shown to cause autosomal dominantly inherited Axenfeld‐Rieger's Syndrome (ARS), a developmental disorder associated in eye anomalies and glaucoma. Interestingly, while FOXC1 missense mutations that cause ARS reduce FOXC1 activity, increased FOXC1 function now appears to be often linked to more aggressive cancer phenotypes in TN/BL‐BC, HCC, HL, and NHL.We have investigated the mechanism(s) by which FOXC1 activity is increased in BLBC. Samples were obtained from TNBC tumors, Triple Positive Breast Cancer (TPBC) tumors or from breast tissue from normal patients. Using quantitative PCR, we found that FOXC1 was significantly over‐expressed in TNBC patients as compared to controls. In contrast, FOXC1 mRNA was significantly less expressed in TPBC samples as compared to either control or TNBC samples. FOXC1 protein half‐life was significantly longer in the TNBC/BLBC cell lines (HS 587T and BT 549) compared to FOXC1 protein's half‐life in HeLa cells. Studies of FOXC1 Copy‐number variation (CNV) in TNBC/BLBC cell lines reveals that cell lines that have higher levels of FOXC1 protein (HS 587T, BT‐549) have extra copies of FOXC1. This contrasts with a cell line with lower expression of FOXC1 protein, MDA‐MB‐231, which may be explained by a deletion of FOXC1 in this TNBC cell line. Our results suggest that increased FOXC1 function in TNBC/BLBC results from over‐expression of FOXC1 in tumors of TNBC patients that appears to be the result of changes to FOXC1 stability and amplification of FOXC1 copy number. We predict that understanding the mechanism(s) underlying FOXC1's activation in cancer could aid in designing improved therapies for TNBC patients.Support or Funding InformationVerag Donation to MAWThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FOXC1, the new player in the cancer sandbox.

In recent years, rapidly accumulating evidence implicates forkhead box C1 (FOXC1) in cancer, especially in studies of basal-like breast cancer (BLBC). Other studies have followed suit, demonstrating that FOXC1 is not only a major player in this breast cancer subtype, but also in hepatocellular carcinoma (HCC), endometrial cancer, Hodgkin's lymphoma (HL), and non-Hodgkin's lymphoma (NHL). The FOXC1 gene encodes a transcription factor that is crucial to mesodermal, neural crest, and ocular development, and mutations found in FOXC1 have been found to cause dominantly inherited Axenfeld-Rieger Syndrome (ARS). Interestingly, while FOXC1 missense mutations that are associated with ARS usually reduce gene activity, increased FOXC1 function now appears to be often linked to more aggressive cancer phenotypes in BLBC, HCC, HL, and NHL. This review discusses not only the role of FOXC1 in cancer cell progression, proliferation, differentiation, and metastasis, but also the underlying mechanisms of how FOXC1 can contribute to aggressive cancer phenotypes.

Comparison of Bioinformatics Prediction, Molecular Modeling, and Functional Analyses of FOXC1 Mutations in Patients with Axenfeld-Rieger Syndrome.

Mutations in the forkhead box C1 gene (FOXC1) cause Axenfeld-Rieger syndrome (ARS). Here, we investigated the effect of four ARS missense variants on FOXC1 structure and function, and examined the predictive value of four in silico programs for all 31 FOXC1 missense variants identified to date. Molecular modeling of the FOXC1 forkhead domain predicts that c.402G> A (p.C135Y) alters FOXC1's structure. In contrast, c.378A> G (p.H128R) and c.481A> G (p.M161V) are not predicted to change FOXC1's structure. Functional analysis indicates that p.H128R reduced DNA binding, transactivation, nuclear localization, and has a longer protein half-life than normal. p.C135Y significantly disrupts FOXC1's DNA binding, transactivation, and nuclear localization. p.M161V reduces transactivation capacity without affecting other FOXC1 functions. C.1103C> A (p.T368N) is indistinguishable from wild-type FOXC1 in all tests, consistent with being a rare benign variant. Comparison of these four variants, plus 18 previously characterized FOXC1 missense variants, with predictions from four commonly used in silico bioinformatics programs indicated that sorting intolerant from tolerant (SIFT), polymorphism phenotyping (PolyPhen-2), and MutPred can sensitively identify as pathogenic only FOXC1 mutations with significant functional defects. This information was used to predict, as disease-causing, nine additional FOXC1 missense variations. Importantly, our results indicate SIFT, PolyPhen-2, and MutPred can reliably be used to predict missense variant pathogenicity for forkhead transcription factors.

Whole exome sequencing reveals a novel de novo FOXC1 mutation in a patient with unrecognized Axenfeld–Rieger syndrome and glaucoma

We report the identification of a novel mutation in the fork-head box C1 (FOXC1) gene which occurred de novo in an Italian patient with unrecognized Axenfeld–Rieger syndrome. He was previously diagnosed as having late recognized primary congenital glaucoma at the age of 14years and was subsequently subjected to multiple surgical interventions due to uncontrolled intraocular pressure and progressive visual field loss.After exclusion of mutations in CYP1B1 and MYOC, trio-whole-exome sequencing revealed de novo in frame deletion in the coding region of the FOXC1 gene (c.407_409delGTC, p.V137del) leading to a deletion of the evolutionary conserved amino acid Valine at position 137 of the protein. Molecular modeling predicted that Val137 deletion impairs FOXC1 DNA-binding capacity and transcriptional activation.Since loss-of-function mutations in FOXC1 are associated with Axenfeld–Rieger syndrome, the genetic findings in combination with re-evaluation of the patient's clinical data resulted in a corrected diagnosis of Axenfeld–Rieger syndrome with developmental glaucoma. We therefore suggest that in addition to CYP1B1 and MYOC, FOXC1 should be included in the genetic analysis of cases with unclear glaucomatous phenotypes to ensure proper diagnosis, adequate treatment and appropriate genetic counseling.

The expression and functional role of a FOXC1 related mRNA-lncRNA pair in oral squamous cell carcinoma.

The Fork head box C1 (FOXC1) gene is overexpressed in multiple malignant tumors and is functionally correlated with tumor progression. However, its’ role in oral squamous cell carcinoma (OSCC) is still unclear. Recent studies have revealed that many long non-coding RNA (lncRNAs) cooperate with adjacent coding genes and form a functional “lncRNA-mRNA pair”. In this study, we report a new lncRNA FOXC1 upstream transcript (FOXCUT) that was remarkably overexpressed in 23 OSCC patients, as was the adjacent FOXC1 gene. The expressions of FOXC1 and FOXCUT were positively correlated. When the expression of FOXCUT was down-regulated by small interfering RNA (siRNA), the expression of FOXC1 was also decreased. Moreover, in OSCC cells Tca8113 and SCC-9, down-regulation of either FOXC1 or FOXCUT by siRNA could inhibit cell proliferation and cell migration in vitro and was accompanied with a reduction of MMP2, MMP7, MMP9, and VEGF-A. In conclusion, FOXC1 may be co-amplified with FOXCUT in OSCC, and both of them may be functionally involved in the tumor progression of OSCC. This provides evidence that both FOXC1 and FOXCUT may serve as novel biomarkers and therapeutic targets in OSCC patients who overexpress this “lncRNA-mRNA pair”.

Human p32 Is a Novel FOXC1-Interacting Protein That Regulates FOXC1 Transcriptional Activity in Ocular Cells

Mutations in the human forkhead box C1 gene (FOXC1) cause Axenfeld-Rieger (AR) malformations, often leading to glaucoma. Understanding the function of FOXC1 necessitates characterizing the proteins that interact with FOXC1. This study was undertaken to isolate FOXC1-interacting proteins and determine their effects on FOXC1. To identify FOXC1-interacting proteins, a human trabecular meshwork (HTM) yeast two-hybrid (Y2H) cDNA library was screened. The interaction and colocalization between FOXC1 and its putative protein partner were confirmed by Ni(2+) pull-down assays, immunoprecipitation, and immunofluorescence, respectively. The electrophoretic mobility shift assay (EMSA) was used to study the effect of the interacting protein on FOXC1 DNA-binding ability. Dual luciferase assays using FOXC1 reporter plasmids in HTM cells were performed to determine the effect of the interaction on FOXC1 transcription activity. The human p32 protein was isolated as a putative FOXC1-interacting protein from a Y2H screen. The interaction of FOXC1 with p32 was confirmed by Ni-pull-down assays and immunoprecipitation. Although p32 is predominantly cytoplasmic, the portion of p32 that is within the nucleus colocalizes with FOXC1. The FOXC1 forkhead domain (FHD) was identified as the p32 interaction domain. p32 significantly inhibited FOXC1-mediated transcription activation in a dose-dependent manner but did not affect FOXC1 DNA-binding ability. Of interest, a FOXC1 mutation F112S displayed an impaired interaction with p32. In the study, the human p32 protein as a novel regulator of FOXC1-mediated transcription activation. Failure of p32 to interact with FOXC1 containing the disease-causing F112S mutation indicates that impaired protein interaction may be a disease mechanism for AR malformations.