Remdesivir ameliorates ulcerative colitis-propelled cell inflammation and pyroptosis in acetic acid rats by restoring SIRT6/FoxC1 pathway.

INTRODUCTION: Ulcerative colitis (UC) is a primary culprit of inflammatory bowel disease that entails prompt and effective clinical intervention. Remdesivir (RDV), a broad-spectrum antiviral nucleotide, has been found to exert anti-inflammatory effects in experimental animals. AIM: This study investigates the prospective anti-inflammatory merit of RDV on an experimental model of UC. The role of SIRT6/FoxC1 in regulating colonic cell inflammation and pyroptosis is delineated. METHOD: Rats were challenged with a single intrarectal dose of acetic acid (AA) solution (2 ml; 4 % v/v) to induce colitis. RDV (20 mg/kg, ip) and sulfasalazine (100 mg/kg, po) were administered to rats 14 days before the injection of AA. RESULTS: Administration of RDV ameliorated colonic cell injury and loss as manifested by improvement of severe colon histopathological mutilation and macroscopic damage and disease activity index scores together with restoration of normal colon weight/length ratio. In addition, RDV alleviated colonic inflammatory reactions, thereby curtailing NF-κB activation and the inflammatory cytokines, TNF-α, IL-18, and IL-1ß. Mitigation of colonic oxidative stress and apoptotic reactions were also evident in the setting of RDV treatment. Mechanistically, RDV enhanced the anti-inflammatory cascade, SIRT6/FoxC1, together with curbing the pyroptotic signal, NLRP3/cleaved caspase-1/Gasdermin D-elicited colonic inflammatory cell death. CONCLUSION: This study reveals, for the first time, the anti-inflammatory effect of RDV against experimental UC. Augmenting SIRT6/FoxC1-mediated repression of colonic inflammation and pyroptosis might advocate the colo-protective potential of RDV.

Characterization of age-associated gene expression changes in mouse sweat glands.

Evaporation of sweat on the skin surface is the major mechanism for dissipating heat in humans. The secretory capacity of sweat glands (SWGs) declines during aging, leading to heat intolerance in the elderly, but the mechanisms responsible for this decline are poorly understood. We investigated the molecular changes accompanying SWG aging in mice, where sweat tests confirmed a significant reduction of active SWGs in old mice relative to young mice. We first identified SWG-enriched mRNAs by comparing the skin transcriptome of Eda mutant Tabby male mice, which lack SWGs, with that of wild-type control mice by RNA-sequencing analysis. This comparison revealed 171 mRNAs enriched in SWGs, including 47 mRNAs encoding 'core secretory' proteins such as transcription factors, ion channels, ion transporters, and trans-synaptic signaling proteins. Among these, 28 SWG-enriched mRNAs showed significantly altered abundance in the aged male footpad skin, and 11 of them, including Foxa1, Best2, Chrm3, and Foxc1 mRNAs, were found in the 'core secretory' category. Consistent with the changes in mRNA expression levels, immunohistology revealed that higher numbers of secretory cells from old SWGs express the transcription factor FOXC1, the protein product of Foxc1 mRNA. In sum, our study identified mRNAs enriched in SWGs, including those that encode core secretory proteins, and altered abundance of these mRNAs and proteins with aging in mouse SWGs.

Variants in FOXC1 and FOXC2 identified in patients with conotruncal heart defects.

Conotruncal heart defects (CTD), subtypes of congenital heart disease, result from abnormal cardiac outflow tract development (OFT). FOXC1 and FOXC2 are closely related members of the forkhead transcription factor family and play essential roles in the development of OFT. We confirmed their expression pattern in mouse and human embryos, identifying four variants in FOXC1 and three in FOXC2 by screening these two genes in 605 patients with sporadic CTD. Western blot demonstrated expression levels, while Dual-luciferase reporter assay revealed affected transcriptional abilities for TBX1 enhancer in two FOXC1 variants and three FOXC2 variants. This might result from the altered DNA-binding abilities of mutant proteins. These results indicate that functionally impaired FOXC1 and FOXC2 variants may contribute to the occurrence of CTD.

Exploring the Genetic Landscape of Childhood Glaucoma.

Childhood glaucoma, a significant cause of global blindness, represents a heterogeneous group of disorders categorized into primary or secondary forms. Primary childhood glaucoma stands as the most prevalent subtype, comprising primary congenital glaucoma (PCG) and juvenile open-angle glaucoma (JOAG). Presently, multiple genes are implicated in inherited forms of primary childhood glaucoma. This comprehensive review delves into genetic investigations into primary childhood glaucoma, with a focus on identifying causative genes, understanding their inheritance patterns, exploring essential biological pathways in disease pathogenesis, and utilizing animal models to study these mechanisms. Specifically, attention is directed towards genes such as CYP1B1 (cytochrome P450 family 1 subfamily B member 1), LTBP2 (latent transforming growth factor beta binding protein 2), TEK (TEK receptor tyrosine kinase), ANGPT1 (angiopoietin 1), and FOXC1 (forkhead box C1), all associated with PCG; and MYOC (myocilin), associated with JOAG. Through exploring these genetic factors, this review aims to deepen our understanding of the intricate pathogenesis of primary childhood glaucoma, thereby facilitating the development of enhanced diagnostic and therapeutic strategies.

Structural Variant Disrupting the Expression of the Remote FOXC1 Gene in a Patient with Syndromic Complex Microphthalmia.

Ocular malformations (OMs) arise from early defects during embryonic eye development. Despite the identification of over 100 genes linked to this heterogeneous group of disorders, the genetic cause remains unknown for half of the individuals following Whole-Exome Sequencing. Diagnosis procedures are further hampered by the difficulty of studying samples from clinically relevant tissue, which is one of the main obstacles in OMs. Whole-Genome Sequencing (WGS) to screen for non-coding regions and structural variants may unveil new diagnoses for OM individuals. In this study, we report a patient exhibiting a syndromic OM with a de novo 3.15 Mb inversion in the 6p25 region identified by WGS. This balanced structural variant was located 100 kb away from the FOXC1 gene, previously associated with ocular defects in the literature. We hypothesized that the inversion disrupts the topologically associating domain of FOXC1 and impairs the expression of the gene. Using a new type of samples to study transcripts, we were able to show that the patient presented monoallelic expression of FOXC1 in conjunctival cells, consistent with the abolition of the expression of the inverted allele. This report underscores the importance of investigating structural variants, even in non-coding regions, in individuals affected by ocular malformations.

FOXC1 Promotes Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells via the Dnmt3b/CXCL12 Axis.

Bone defects have remained a clinical problem in current orthopedics. Bone marrow mesenchymal stem cells (BM-MSCs) with multi-directional differentiation ability have become a research hotspot for repairing bone defects. In vitro and in vivo models were constructed, respectively. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to detect osteogenic differentiation ability. Western blotting (WB) was used to detect the expression of osteogenic differentiation-related proteins. Serum inflammatory cytokine levels were detected by ELISA. Fracture recovery was evaluated by HE staining. The binding relationship between FOXC1 and Dnmt3b was verified by dual-luciferase reporter assay. The relationship between Dnmt3b and CXCL12 was explored by MSP and ChIP assays. FOXC1 overexpression promoted calcium nodule formation, upregulated osteogenic differentiation-related protein expression, promoted osteogenic differentiation, and decreased inflammatory factor levels in BM-MSCs, and promoted callus formation, upregulated osteogenic differentiation-related protein expression, and downregulated CXCL12 expression in the mouse model. Furthermore, FOXC1 targeted Dnmt3b, with Dnmt3b knockdown decreasing calcium nodule formation and downregulating osteogenic differentiation-related protein expression. Additionally, inhibiting Dnmt3b expression upregulated CXCL12 protein expression and inhibited CXCL12 methylation. Dnmt3b could be binded to CXCL12. CXCL12 overexpression attenuated the effects of FOXC1 overexpression and inhibited BM-MSCs osteogenic differentiation. This study confirmed that the FOXC1-mediated regulation of the Dnmt3b/CXCL12 axis had positive effects on the osteogenic differentiation of BM-MSCs.

Research progress on the regulatory mechanisms of FOXC1 expression in cancers and its role in drug resistance.

The Forkhead box C1 (FOXC1) transcription factor is an important member of the FOX family. After initially being identified in triple-negative breast cancer (TNBC) with significant oncogenic function, FOXC1 was subsequently demonstrated to be involved in the development of more than 16 types of cancers. In recent years, increasing studies have focused on the deregulatory mechanisms of FOXC1 expression and revealed that FOXC1 expression was regulated at multiple levels including transcriptional regulation, post-transcription regulation and post-translational modification. Moreover, dysregulation of FOXC1 is also implicated in drug resistance in various types of cancer, especially in breast cancer, which further emphasizes the translational and clinical significance of FOXC1 as a therapeutic target in cancer treatment. This review summarizes recent findings on mechanisms of FOXC1 dysregulation in cancers and its role in chemoresistance, which will help to better understand the oncogenic role of FOXC1, overcome FOXC1-mediated drug resistance and develop targeted therapy for FOXC1 in cancers.

Isorhynchophylline attenuates proliferation and migration of synovial fibroblasts via the FOXC1/ß-catenin axis.

Rheumatoid arthritis (RA) is a common type of chronic inflammatory disease. Elucidating the mechanism of fibroblast-like synovial (FLS) as a pathologic factor in RA may address the urgent medical requirement for the treatment of RA. Isorhynchophylline (IRN) is a tetracyclic hydroxyindole alkaloid isolated from uncinaria, which has multiple biological activities and affects the progression of osteoarthritis. However, the role of IRN in rheumatoid arthritis remains unclear. Herein, our study aimed to elucidate the potential effect of IRN on RA and reveal its mechanism. Human FLS cell line MH7A cells were stimulated with TNF-α for 24 h to construct a cell model. CCK-8, Edu, wound healing, as well as transwell assays were conducted to detect the effects of IRN on cell proliferation and motility. ELISA and Immunoblot assays were further performed to detect the production of pro-inflammatory factors and the expression levels of MMPs. Immunoblot and Immunostaining assays were conducted to uncover the mechanism. ELISA, H&E staining, and Immunoblot assays were used to confirm the effects of IRN on RA in a CIA rat model. We revealed that IRN restrained TNF-α-stimulated MH7A cell proliferation and motility. In addition, IRN blocked the production of pro-inflammatory factors and MMPs in TNF-α-stimulated-MH7A cells. We further found that IRN restrained FOXC1/ß-catenin axis, and improved MH7A cell proliferation as well as migration via the FOXC1/ß-catenin axis. IRN restores CIA by inhibiting pro-inflammatory cytokines in synovial tissues. In summary, IRN attenuates proliferation and migration of FLS in RA via the FOXC1 mediated ß-catenin axis.

Icaritin inhibits endometrial carcinoma cells by suppressing O-GlcNAcylation of FOXC1.

BACKGROUND: Icaritin has a wide range of pharmacological activities, including significant an-titumor activity. However, the mechanism of action of icaritin in endometrial cancer (UCEC) remains unknown. FOX proteins are a highly conserved transcription factor superfamily that play important roles in epithelial cell differentiation, tumor metastasis, angiogenesis, and cell cycle regulation. FOXC1 is an important member of the FOX protein family. FOXC1 is aberrantly expressed in endometrial cancer and may play a role in the migration and invasion of endometrial cancer; however, its mechanism of action has not yet been reported. O-GlcNAc glycosylation is a common post-translational modification. In endometrial cancer, high levels of O-GlcNAcylation promote cell proliferation, migration, and invasion. Cancer development is often accompanied by O-GlcNAc modification of proteins; however, O-GlcNAc modification of the transcription factor FOXC1 has not been reported to date. PURPOSE: To investigate the inhibitory effects of icaritin on RL95-2 and Ishikawa endometrial cancer cells in vitro and in vivo and to elucidate the possible molecular mechanisms. METHODS/STUDY DESIGN: CCK8, colony formation, migration, and invasion assays were used to determine the inhibitory effects of icaritin on endometrial cancer cells in vitro. Cell cycle regulation was assayed by flow cytometry. Protein levels were measured based on western blotting. The level of FOXC1 expression in endometrial cancer tissues was determined by immunohistochemistry. To assess whether icaritin also has activity in vivo, its effect on tumor xenografts was evaluated. RESULTS: Immunohistochemical analysis of clinical samples revealed that FOXC1 expression was significantly higher in endometrial cancer tissues than in normal tissues. Downregulation of FOXC1 inhibited the proliferative, colony formation, migration, and invasive abilities of RL95-2 and Ishikawa endometrial cancer cells. Icaritin inhibited the proliferation, colony formation, migration, and invasion of endometrial cancer cells and blocked the cell cycle in S phase. Icaritin affected O-GlcNAc modification of FOXC1 and thus the stability of FOXC1, which subsequently triggered the inhibition of endometrial cancer cell proliferation. CONCLUSION: The anti-endometrial cancer effect of icaritin is related to the inhibition of abnormal O-GlcNAc modification of FOXC1, which may provide an important theoretical foundation for the use of icaritin against endometrial cancer.

FOXC1 and FOXC2 maintain mitral valve endothelial cell junctions, extracellular matrix, and lymphatic vessels to prevent myxomatous degeneration.

Background: Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates mutations of the transcription factor FOXC1 are associated with MV defects, including mitral valve regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2, are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar extracellular matrix (ECM). Methods: Adult mice carrying tamoxifen-inducible, endothelial cell (EC)-specific, compound Foxc1;Foxc2 mutations (i.e., EC-Foxc-DKO mice) were used to study the function of Foxc1 and Foxc2 in the maintenance of mitral valves. The EC-mutations of Foxc1/c2 were induced at 7 - 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of EC-Foxc-DKO mice were assessed via whole-mount immunostaining, immunohistochemistry, and Movat pentachrome/Masson's Trichrome staining. Results: EC-deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker mitral valves by causing defects in regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC-Foxc1/c2 mutant mice. PROX1, a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC-Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded in EC-Foxc1/c2 mutant mitral valves. Conclusions: Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessels to prevent myxomatous mitral valve degeneration.

Regulation and Function of FOXC1 in Osteoblasts.

Estrogens, which bind to estrogen receptor alpha (ERα), are important for proper bone mineral density. When women go through menopause, estrogen levels decrease, and there is a decrease in bone quality, along with an increased risk for fractures. We previously identified an enhancer near FOXC1 as the most significantly enriched binding site for estrogen receptor alpha (ERα) in osteoblasts. FOXC1 is a transcription factor belonging to a large group of proteins known as forkhead box genes and is an important regulator of bone formation. Here, we demonstrate that 17ß-estradiol (E2) increases the mRNA and protein levels of FOXC1 in primary mouse and human osteoblasts. GATA4 is a pioneer factor for ERα and it is also recruited to enhancers near Foxc1. Knockdown of Gata4 in mouse osteoblasts in vitro decreases Foxc1 expression as does knockout of Gata4 in vivo. Functionally, GATA4 and FOXC1 interact and regulate osteoblast proteins such as RUNX2, as demonstrated by ChIP-reChIP and luciferase assays. The most enriched motif in GATA4 binding sites from ChIP-seq is for FOXC1, supporting the notion that GATA4 and FOXC1 cooperate in regulating osteoblast differentiation. Together, these data demonstrate the interactions of the transcription factors ERα, GATA4, and FOXC1 to regulate each other's expression and other osteoblast differentiation genes.

Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Single-cell RNA sequencing reveals tumor heterogeneity within salivary gland pleomorphic adenoma: A preliminary study.

BACKGROUND: Salivary gland pleomorphic adenoma (SPA) is a common neoplasm of salivary glands that displays remarkable histological diversity. Previous studies have demonstrated the involvement of gene rearrangements and cytoskeleton-remodeling-related myoepithelial cells in SPA tumorigenesis. Cytoskeleton remodeling is necessary for epithelial-mesenchymal transition (EMT), a key process in tumor progression. However, the heterogeneity of tumor cells and cytoskeleton remodeling in SPA has not been extensively investigated. METHODS: An analysis of single-cell RNA sequencing (scRNA-seq) was performed on 27 810 cells from two donors with SPA. Bioinformatic tools were used to assess differentially expressed genes, cell trajectories, and intercellular communications. Immunohistochemistry and double immunofluorescence staining were used to demonstrate FOXC1 and MYLK expression in SPA tissues. RESULTS: Our analysis revealed five distinct cell subtypes within the tumor cells of SPA, indicating a high level of intra-lesional heterogeneity. Cytoskeleton-remodeling-related genes were highly enriched in subtype 3 of the tumor cells, which showed a close interaction with mesenchymal cells. We found that tumoral FOXC1 expression was closely related to MYLK expression in the tumor cells of SPA. CONCLUSION: Tumor cells enriched with cytoskeleton-remodeling-related genes play a crucial role in SPA development, and FOXC1 may partially regulate this process.

N-3, a novel synthetic derivative of bifendate, inhibits metastasis of triple-negative breast cancer via decreasing p38-regulated FOXC1 protein stability.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with high invasiveness, metastatic potential, and poor prognosis. Epithelial-mesenchymal transition (EMT) is pivotal in TNBC progression, becoming a promising target for TNBC treatment. Our study evaluated N-3, a novel synthetic bifendate derivative, which inhibited the EMT-associated migration and invasion of MDA-MB-231 and 4T1 TNBC cells. The results were consistent with the suppression of FOXC1 expression and transcriptional activity. Additional studies indicated that N-3 reduced the protein stability of FOXC1 by enhancing ubiquitination and degradation. Moreover, N-3 downregulated p-p38 expression and FOXC1 interaction, decreasing the stability of p38-regulated FOXC1. Further, N-3 blocked TNBC metastasis with an artificial lung metastasis model in vivo, related to FOXC1 suppression and EMT. These results highlight the potential of N-3 as a TNBC metastasis treatment. Therefore, FOXC1 regulation could be a novel targeted therapeutic strategy for TNBC metastasis.

Case report: Extending the spectrum of clinical and molecular findings in FOXC1 haploinsufficiency syndrome.

FOXC1 is a ubiquitously expressed forkhead transcription factor that plays a critical role during early development. Germline pathogenic variants in FOXC1 are associated with anterior segment dysgenesis and Axenfeld-Rieger syndrome (ARS, #602482), an autosomal dominant condition with ophthalmologic anterior segment abnormalities, high risk for glaucoma and extraocular findings including distinctive facial features, as well as dental, skeletal, audiologic, and cardiac anomalies. De Hauwere syndrome is an ultrarare condition previously associated with 6p microdeletions and characterized by anterior segment dysgenesis, joint instability, short stature, hydrocephalus, and skeletal abnormalities. Here, we report clinical findings of two unrelated adult females with FOXC1 haploinsufficiency who have ARS and skeletal abnormalities. Final molecular diagnoses of both patients were achieved using genome sequencing. Patient 1 had a complex rearrangement involving a 4.9 kB deletion including FOXC1 coding region (Hg19; chr6:1,609,721-1,614,709), as well as a 7 MB inversion (Hg19; chr6:1,614,710-8,676,899) and a second deletion of 7.1 kb (Hg19; chr6:8,676,900-8,684,071). Patient 2 had a heterozygous single nucleotide deletion, resulting in a frameshift and a premature stop codon in FOXC1 (NM_001453.3): c.467del, p.(Pro156Argfs*25). Both individuals had moderate short stature, skeletal abnormalities, anterior segment dysgenesis, glaucoma, joint laxity, pes planovalgus, dental anomalies, hydrocephalus, distinctive facial features, and normal intelligence. Skeletal surveys revealed dolichospondyly, epiphyseal hypoplasia of femoral and humeral heads, dolichocephaly with frontal bossin gand gracile long bones. We conclude that haploinsufficiency of FOXC1 causes ARS and a broad spectrum of symptoms with variable expressivity that at its most severe end also includes a phenotype overlapping with De Hauwere syndrome.

Primary open-angle glaucoma risk prediction with ABCA1 and LOC102723944 variants and their genotype-phenotype correlations in southern Chinese population.

Glaucoma is a leading cause of irreversible visual impairment and blindness worldwide. Previous genome-wide association studies have identified caveolin-1 (CAV1), ATP-binding cassette A1 (ABCA1), and forkhead box C1 (FOXC1) loci associated with primary open angle glaucoma (POAG), a major subtype of glaucoma. This study aimed to fine map the association pattern of FOXC1 locus with POAG and determine the correlations of FOXC1, ABCA1, and CAV1 variants with ocular and lipidemic parameters in southern Chinese population. In total, 1291 unrelated Han Chinese subjects were recruited, including 301 high-tension glaucoma (HTG), 126 normal-tension glaucoma (NTG), and 864 control subjects. Twelve variants in FOXC1 locus, and two variants in ABCA1 and CAV1 genes, were genotyped by TaqMan assays. Genetic risk score and genotype-phenotype correlation analyses were conducted. In the FOXC1 locus, LOC102723944 rs6596830, rather than previously reported rs2745572, showed significant association with POAG (P = 8.61 × 10-4, odds ratio (OR) = 0.75) and HTG (P = 3.68 × 10-3, OR = 0.75). ABCA1 rs2487032 was also significantly associated with POAG (P = 3.00 × 10-5, OR = 0.70) and HTG (P = 2.08 × 10-4, OR = 0.70). Joint analysis showed that carriers of homozygous non-protective alleles of ABCA1 rs2487032 and LOC102723944 rs6596830 had 2.99-fold higher risk of POAG (P = 1.27 × 10-3) when compared to those carrying homozygous non-risk alleles. Patients with POAG carrying ABCA1 rs2487032 G allele had higher HDL cholesterol, and those with LOC102723944 rs6596830 A allele had lower LDL. This study revealed individual and joint association of ABCA1 and LOC102723944 variants with POAG in southern Chinese population. Subjects carrying non-protective alleles had increased risk to POAG, and corresponding genotypes would affect the lipid profiles.

FoxC1 activates limbal epithelial stem cells following corneal epithelial debridement.

Limbal epithelial stem cells are not only critical for corneal epithelial homeostasis but also have the capacity to change from a relatively quiescent mitotic phenotype to a rapidly proliferating cell in response to population depletion following corneal epithelial wounding. Pax6+/- mice display many abnormalities including corneal vascularization and these aberrations are consistent with a limbal stem cell deficiency (LSCD) phenotype. FoxC1 has an inhibitory effect on corneal avascularity and a positive role in stem cell maintenance in many tissues. However, the role of FoxC1 in limbal epithelial stem cells remains unknown. To unravel FoxC1's role(s) in limbal epithelial stem cell homeostasis, we utilized an adeno-associated virus (AAV) vector to topically deliver human FOXC1 proteins into Pax6 +/- mouse limbal epithelium. Under unperturbed conditions, overexpression of FOXC1 in the limbal epithelium had little significant change in differentiation (PAI-2, Krt12) and proliferation (BrdU, Ki67). Conversely, such overexpression resulted in a marked increase in the expression of putative limbal epithelial stem cell markers, N-cadherin and Lrig1. After corneal injuries in Pax6 +/- mice, FOXC1 overexpression enhanced the behavior of limbal epithelial stem cells from quiescence to a highly proliferative status. Overall, the treatment of AAV8-FOXC1 may be beneficial to the function of limbal epithelial stem cells in the context of a deficiency of Pax6 function.

Spectrum of white matter abnormalities associated with FOXC1-related disorders in two unrelated cases.

The purpose of this study is to document the wide spectrum of white matter abnormalities associated with FOXC1 pathogenic variants. We report two adult individuals-a 60-year-old individual and a 24-year-old one, presenting with hearing loss, anterior eye segment dysgenesis, and very different severity of cerebral small vessel disease. Molecular testing documented the presence of FOXC1 pathogenic variants in both individuals. Our paper documents the broad spectrum of radiological white matter involvement in adult individuals with FOXC1-related disorders. Mild forms of FOXC1-related small vessel disease, as we observed in individual 2, should be included in the list of genetic mimickers of MS.

Foxc1 and Foxc2 function in osteochondral progenitors for the progression through chondrocyte hypertrophy and mineralization of the primary ossification center.

The forkhead box transcription factor genes Foxc1 and Foxc2 are expressed in the condensing mesenchyme of the developing skeleton prior to the onset of chondrocyte differentiation. To determine the roles of these transcription factors in limb development we deleted both Foxc1 and Foxc2 in lateral plate mesoderm using the Prx1-cre mouse line. Resulting compound homozygous mice died shortly after birth with exencephaly, and malformations to this sternum and limb skeleton. Notably distal limb structures were preferentially affected, with the autopods displaying reduced or absent mineralization. The radius and tibia bowed and the ulna and fibula were reduced to an unmineralized rudimentary structure. Molecular analysis revealed reduced expression of Ihh leading to reduced proliferation and delayed chondrocyte hypertrophy at E14.5. At later ages, Prx1-cre;Foxc1Δ/ Δ;Foxc2 Δ / Δ embryos exhibited restored Ihh expression and an expanded COLX-positive hypertrophic chondrocyte region, indicating a delayed exit and impaired remodeling of the hypertrophic chondrocytes. Osteoblast differentiation and mineralization were disrupted at the osteochondral junction and in the primary ossification center (POC). Levels of OSTEOPONTIN were elevated in the POC of compound homozygous mutants, while expression of Phex was reduced, indicating that impaired OPN processing by PHEX may underlie the mineralization defect we observe. Together our findings suggest that Foxc1 and Foxc2 act at different stages of endochondral ossification. Initially these genes act during the onset of chondrogenesis leading to the formation of hypertrophic chondrocytes. At later stages Foxc1 and Foxc2 are required for remodeling of HC and for Phex expression required for mineralization of the POC.

Analysis of glaucoma genes in Finnish patients with juvenile open-angle glaucoma.

PURPOSE: To identify germline variants in myocilin (MYOC) and other known monogenic glaucoma genes in Finnish patients with juvenile open-angle glaucoma (JOAG). METHODS: Finnish patients with JOAG treated between 2010 and 2018 at the Department of Ophthalmology, Helsinki University Hospital, Finland, were enrolled. We sequenced all exonic regions and flanking splice sites of MYOC for five patients and one healthy relative using Sanger sequencing. In 48 patients, we performed exome sequencing to identify variants also in 28 other glaucoma-related genes. RESULTS: Fifty-three individuals with JOAG from 50 pedigrees, and one healthy relative, participated. The mean age at diagnosis was 30.8 years [SD 7.6; range 11 to 39]. Five probands had probably pathogenic variants in MYOC: c.1102C>T p.(Gln368Ter), c.1109C>T p.(Pro370Leu), c.1130C>T p.(Thr377Met), c.1132G>A p.(Asp378Asn) and c.1456C>T p.(Leu486Phe). Four of these patients had a family history of dominantly inherited JOAG. The frequency of MYOC variants was 10% (5 of 50 families). One patient and his mother with JOAG had a novel loss-of-function variant in the FOXC1 gene, c.366G>A p.(Trp122Ter). A patient with sporadic JOAG had a homozygous likely pathogenic variant in the LTBP2 gene, c.3938G>A p.(Cys1313Tyr). The genetic variants explained 14% (7 out of 50 families; 95% CI, 6%-23%) of JOAG in our cohort. CONCLUSIONS: The frequency of pathogenic variants in previously known glaucoma-associated genes is low in Finnish patients with JOAG. Because of the distinct genetic background of Finns, it might be possible to identify novel glaucoma genes through our JOAG series in the future.