Abstract
Forkhead-box C2 (FOXC2) is a transcription factor involved in lymphatic system development. FOXC2 mutations cause Lymphedema-distichiasis syndrome (LD). Recently, a natural antisense was identified, called lncRNA FOXC2-AS1, which increases FOXC2 mRNA stability. No studies have evaluated FOXC2 and FOXC2-AS1 blood expression in LD and healthy subjects. Here, we show that FOXC2 and FOXC-AS1 expression levels were similar in both controls and patients, and a significantly higher amount of both RNAs was observed in females. A positive correlation between FOXC2 and FOXC2-AS1 expression was found in both controls and patients, excluding those with frameshift mutations. In these patients, the FOXC2-AS1/FOXC2 ratio was about 1:1, while it was higher in controls and patients carrying other types of mutations. The overexpression or silencing of FOXC2-AS1 determined a significant increase or reduction in FOXC2 wild-type and frameshift mutant proteins, respectively. Moreover, confocal and bioinformatic analysis revealed that these variations caused the formation of nuclear proteins aggregates also involving DNA. In conclusion, patients with frameshift mutations presented lower values of the FOXC2-AS1/FOXC2 ratio, due to a decrease in FOXC2-AS1 expression. The imbalance between FOXC2 mRNA and its lncRNA could represent a molecular mechanism to reduce the amount of FOXC2 misfolded proteins, protecting cells from damage.
Highlights
Lymphedema-distichiasis syndrome (LD, OMIM 153400) is an autosomal dominant rare disorder in which the lymphatic system, responsible for the production and transport of fluids and immune cells throughout the body, does not develop correctly [1]
The patients enrolled in this study presented different Forkhead-box C2 (FOXC2) variants: three missense mutations (p.I213V, p.V228M and p.A492V), a nonsense mutation (p.Y109*), and two frameshift mutations (p.H199Pfs264* and p.I213Tfs18*)
To evaluate the expression levels of FOXC2 and FOXC2-AS1, total RNA obtained from peripheral blood of healthy subjects, LD patients and HeLa cells was analyzed using Quantitative real time PCR (qRT-PCR) assay (Figure 2A)
Summary
Lymphedema-distichiasis syndrome (LD, OMIM 153400) is an autosomal dominant rare disorder in which the lymphatic system, responsible for the production and transport of fluids and immune cells throughout the body, does not develop correctly [1]. LD is characterized by the swelling of the limbs, in particular the legs and feet, and by the presence of extra eyelashes (distichiasis) on the inner lining of both the upper and lower eyelids. Swollen and knotted veins, drooping eyelids (ptosis), cardiac anomalies, and cleft palate can be observed [2]. FOXC2 is located on the long arm of chromosome 16, positive strand, and consists of a single exon. It encodes a transcription factor (FOXC2), composed of 501 amino acids, regulating the development of some systems during embryogenesis, the lymphatic and blood vascular system. There are some phosphorylation and SUMOylation sites in the central part of the protein (residues 163–394) [7,8]
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