Investigação de novos mecanismos genéticos e epigenéticos relacionados à Síndrome do X-frágil

Abstract

Fragile X syndrome (SXF) is determined by an unstable expansion of CGG trinucleotides repetition in the 5'UTR region of the FMR1 gene, with four recognized allele categories: normal (N, ≤44 CGGs), intermediate (I, 45-54 CGGs), premutation (PM, 55-200 CGGs) and full mutation (FM, > 200 CGGs). SXF patients carry the FM allele, which results in gene silencing by hypermethylation of the CGG repeat and FMR1 gene promoter. CGG expansions instability can result in size mosaicism, formed by FM along with other allele categories or deletions. Due to the difficulty in amplifying this sequence, the molecular investigation of SXF is complex. Besides that, a recent study has suggested a methylation boundary upstream of the CGG repeat, which is lost in SXF patients and leads to a de novo methylation spreading to the FMR1 promoter. Thus, the present study aimed to reevaluate a group of 101 SXF males, to characterize atypical profiles, analyze the presence of the methylation boundary and identify novel epigenetic markers close to the CGG repeat. Using Triplet Repeat Primed Polymerase Chain Reaction (TRP-PCR), a “pure” FM profile was confirmed in 52.5% of the SXF patients. 27.7% of the patients had size mosaicism: 15.8% with PM+FM, 8.9% with N+FM and 3% with N+PM+FM. Six patients (5.9%) had a deletion close to the repetitions, evidenced by additional methodologies, and microhomology could be observed between the breakpoints in some of them. The analysis of genetic markers in X chromosome revealed no cases of Klinefelter syndrome (47,XXY) among the atypical cases. Size mosaicism cases have been reported in the literature with frequencies of 12 to 41%. Using pyrosequencing after genomic DNA treatment with sodium bisulfite, it was possible to observe a reduction in methylation levels in CpGs 65-70 upstream of FMR1 in controls, ratifying the presence of the methylation boundary, absent in FM patients, which corroborates literature data. This boundary also seems to be lost in individuals with size mosaicism. As a highlight of this study, we identified an intronic CpG with an unexpected methylation pattern in the heatmap, being hypermethylated in controls and hypomethylated in FXS patients. Recent studies suggest that the methylation of intragenic elements is involved in the regulation of gene expression. Therefore, this CpG has great potential to be used as a new epigenetic marker for FXS, with a cut-off value of 69.5% and specificity, sensitivity and accuracy of 100%. These promising results may stimulate the development of a new diagnostic methodology for FXS