Triagem de mutações no gene FOXC2 em uma família com a síndrome Linfedema Distiquíase

Abstract

The Lymphedema-Distichiasis Syndrome (LDS) is a developmental disorder of the lymphatic system, which causes edema in several regions of the body and distichiasis, that is, the birth of extra rows of eyelashes originated from the Meibomian gland or in the upper eyelid. Cleft palate, congenital heart and eye malformations may also appear. The disease is monogenic, with an autosomal dominant inheritance pattern, caused by a mutation in the Forkhead Box Protein C2 (FOXC2) gene. In the present study, screening for mutations in the FOXC2 gene was performed in affected members of a family (child, mother and maternal aunt) with a clinical diagnosis of LDS. Mutation screening was done using the Sanger sequencing of PCR amplicons representing the upstrem region, the 5'-UTR, coding region, and part of the 3'-UTR. In addition, the amplicons covering upstrem and downstream of the coding region of the gene were cloned using TOPO TA Cloning Kit and sequenced. No mutations were found in the protein coding region. However, two mutations were observed, one in the promoter and another in the 5'-UTR gene, by sequencing cloned PCR products, these mutations were detected to be in trans. The first one is a previously described polymorphism (rs34221221), which causes a cytosine to thymine substitution at position -512 (NM_005251.3(FOXC2):c.-512C>T). This mutation is located in the promoter and affects a nucleotide four positions upstrem the transcription start site. In the literature, it has already been shown experimentally that this mutation increases the expression of both mRNA and protein in patients with LDS. The second mutation (NM_005251.3(FOXC2):c.-150C>T) was identified for the first time in this study. As both of these mutations map upstrem to FOXC2 coding region, the first effort was to identify FOXC2 promoter, which has not been described in the literature or included in any databases. Using G2.Promoter, it was possible to identify an atypical promoter, with 1101 base pairs, which extends 1000 bp upstrem the transcription start site and downstream in the 5'-UTR. Typical promoter elements (TATA box, CAAT box) were not detected, but a GC box was abolished by the NM_005251.3(FOXC2):c.-150C>T mutation. It was also predicted that this mutation abolished a G-quadruplex, whose score, although high, does not exceed the threshold established by the program used (G4Hunter). This mutation also abolishes the binding sites of some transcription factors (ETV5, ZNF638, TFAP2A, and nGRE) and creates a new one (ETV4). This set of changes suggests that (NM_005251.3(FOXC2):c.-150C>T) is a pathogenic mutation, although based on the data that is available so far, it will not be possible to assess if there is an increase or change in FOXC2 expression. These mutations also affect the FOXC2-AS1 intron, but we were not able to identify specific evidence of functional impact for this transcript. The findings of this study reinforce the necessity to include upstrem and downstream regions of genes in mutation screening studies in monogenic disorders.