Caracterização de variações no número de cópias e definição do estado de metilação nos genes FMR1 e AFF2 pela técnica de MS-MLPA em homens com deficiência intelectual ligada ao cromossomo X

Abstract

Intellectual disability (ID) is characterized by intellectual impairment identified before 18 years of age, accompained by limitations in one or more adaptive skills. The prevalence of ID is estimated to be 2-3% among the general population and a number of cases remains undiagnosed. ID is more commom among males than in females. One explanation for this is the concentration of genes important for cognitive ability along the chromosome X. Mutations in these genes lead to X-linked ID (XLID). The most frequent form of syndromic XLID is Fragile X Syndrome (FXS), characterized by an unstable CGG trinucleotide expansion on the exon 1 of the Fragile X Mental Retardation 1 gene (FMR1). In FXS individuals, the repeat number exceeds 200 (full mutations), leading to epigenetic silencing of the gene due to DNA hipermethylation and chromatin remodeling. Similar to what occurs with the FMR1 gene, a dynamic mutation on the AF4/FMR2 family, member 2 gene (AFF2) involving CCG trinucleotide repeats, is associated to a less frequent and non-syndromic ID, the FRAXE ID. Recent studies have reported that copy number variations (CNVs) may also be involved in the etiology of FXS and FRAXE ID. In this context, the present study aimed to characterize CNVs in the FMR1 and AFF2 genes and define the methylation status of these genes by Methylation Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA). This methodology allows the relative quantification of 45 sequences in a single experiment. For this purpose, DNA samples from 134 males with suggestive XLID were selected from a group of more than 1.100 individuals with ID. Among our study samples, we included nine patients who obtained inconclusive results for the initial trinucleotide repeat expansion screening by polymerase chain reaction (PCR) in the FMR1 gene and four patients with previously detected deletions involving the trinucleotide repeats in the FMR1 gene and two patients with previously detected deletions in the AFF2 gene. One patient with a deletion comprising exon 1 and intron 1 of the FMR1 gene and methylation indicative of the presence of a full mutation has been identified, being classified as a somatic mosaicism case. Additional experiments were performed to determine the deletion breakpoints, but the results obtained were normal, which would be due to the interference of the full mutated allele. The nine inconclusive patients were methylated, pointing to the presence of a full mutation. The four patients with previously detected deletions on the FMR1 gene were methylated, being also classified as somatic mosaicism cases. The remaining patients did not carry any CNVs or abnormal methylation pattern in any of the two genes studied. These results highlight the importance of implementing additional methodologies for molecular diagnosis of FXS and for differential therapeutic purposes in cases of mosaicism. CNVs in the FMR1 and AFF2 genes are rare, but their screening in patients negative for trinucleotide expansions is strongly recommended, in order to define new molecular mechanisms related to these conditions.