Abstract
Methylation at critical CpG sites on the expanded FMR1 gene is crucial for pathological manifestation of fragile X syndrome and fragile X-related disorders. Methylation status from blood, oral mucosa and root hair was analyzed with the FMR1 mPCR kit (Asuragen). Differential allele expression was studied by TP-PCR. Psychological and neurological explorations were performed in the probands. Patient II-1 of family 1 showed an extremely skewed X-chromosome inactivation of the normal allele in blood, oral mucosa cells and root hair. Analysis of differential expression of both alleles in blood showed the preferential expression of the expanded allele. Similarly, patient II-3 of family 2 showed an extremely skewed X-chromosome inactivation of the normal allele in blood, oral mucosa and root hair. Both females presented clinical features compatible with their skewed methylation toward the normal allele. Methylation analysis at critical CpG sites in the first FMR1 exon may predict clinical manifestations in carriers of premutation or full mutation. Analysis of differential expression of both alleles in women using TP-PCR could contribute to clarify the real impact of skewed methylation on the phenotype.
Highlights
Fragile X syndrome (FXS) was the first pathology in which a dynamic mutation was described as the cause of the disease
Phenotypes in women are highly variable and it is generally accepted that skewed X chromosome inactivation (XCI) should have a predominant role no clear correlation has been observed between clinical findings and the methylation analysis obtained by Southern blot (SB) [1518]
Patient II-1 of family 1 (AR of 0,23 to 0 in blood) expressed preferentially the premutated allele and she presented a more severe phenotype than expected for a woman of her age (FXPOI at the age of 34 and neurological symptoms started at the age of 47) that could be related to the toxic effect of the expanded FMR1 messenger
Summary
Fragile X syndrome (FXS) was the first pathology in which a dynamic mutation was described as the cause of the disease. The FMR1 gene has a variable number of CGG repeats in the 5 ́- UTR divided in different categories depending on the number of repeats and its phenotypic correlation: normal from 5 to 44, intermediate from 45 to 54, premutated from 55 to 200 and fully mutated above 200 repeats. For risk assessment of FXS and related disorders the number of repeats and methylation status of the gene should be analysed [1,2]. Alleles with more than 200 repeats are commonly methylated and as a consequence the gene is silenced, which results in the absence of FMRP and the manifestation of the fragile X syndrome, the most common cause of inherited mental disability and autism [3]. We report the methylation status of two CpG sites in the first FMR1 exon using the Amplidex mPCR kit (Asuragen) in normal, pre-mutated and mutated alleles from different tissues in two fragile X families
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