Screening And Functional Research For Pathogenic Mutation Of FOXC2 Gene In Lymphedema-distichiasis Syndrome

Lymphedema-distichiasis syndrome is an autosomal dominant genetic disease caused by mutations in the FOXC2 gene.It is a rare developmental disorder that affects the formation of the lymphatic system,and the main symptoms are chronic lymphedema of the lower limbs and distichiasis.Although lymphedema is not fatal,it seriously affects the health of the body and the aesthetics of the limbs,and it can cause disability in severe cases.Distichiasis manifests as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids,these extra eyelashes tend to tilt backwards,scratch the cornea,cause corneal irritation and tears,and even cause corneal epithelial damage and infection to form corneal ulcers,which can affect vision to varying degrees.In this study,two pedigrees?2263 and 2355?of Lymphedema-distichiasis syndrome were collected,the pathogenic mutation of FOXC2 was found and its function was studied.In the 2263 pedigree,a frameshift mutation caused by the insertion of c.936₉37insCGCCGCC?p.Tyr313ArgfsX152?was found of the FOXC2 gene,this mutation has also been reported in a family of LDS in Europe.A new frameshift mutation of c.937₉38insGCCGCCT?p.Tyr313CysfsX152?was found in the 2355 pedigree.At present,there are few studies on the function of FOXC2 pathogenic mutation,and in order to study the effect of mutations in different domains of FOXC2 protein for protein function,six mutations?Ile85Asn,Tyr99 X,Asn118Lys,Lys132 Glu,Ser370Thr and Gln444Arg?were screened from the case reports of lymphedema and/or distichiasis caused by FOXC2 gene mutation.RT-qPCR,subcellular localization,and dual luciferase were used to study the expression,cell localization and transcriptional activity of eight FOXC2 mutant proteins in vitro.Due to the frameshift mutation,the mutant proteins of both families lost the important C-terminal domain and protein phosphorylation modifications.These two mutant proteins could be located in the nucleus,but the chromatin shrunk,and the DNA-binding activity and transcriptional activity were weakened.Four pathogenic mutations?Ile85Asn,Tyr99 X,Asn118Lys and Lys132Glu?derived from LDS families with mutation sites in FHD changed the combination way of the original amino acid residues with amino acid residues and DNA,and the expression of mutant proteins was decreased and could not be located in the nucleus,the Ile85 Asn and Lys132 Glu mutant proteins also aggregated in the nucleus,the transcriptional activation of four mutant proteins was weakened.The subcellular localization and transcriptional activation of Ser370 Thr mutant protein were not different from wild-type protein,but its protein expression increased.The subcellular localization of Gln444 Arg was the same as wild type,but its protein expression and transcriptional activation ability were increased,which is a function acquired mutation.Through further functional research,we found that LDS are not only related to the change of FOXC2 mutant protein expression,cell localization and transcriptional activation,but also may be related to the down regulation of ANGPT2 and RASAL3 gene.