Abstract
Background: The variable phenotype in female carriers of a full mutation is explained in part by non-random X-chromosome inactivation. The molecular diagnosis of fragile X syndrome is based on the resolution of the number of CGG triplet repeats and the methylation status of a critical CpG in the fragile X mental retardation gene (FMR1) promoter. Neighboring CpGs in the FMR1 promoter are supposed to be equally methylated or unmethylated.
Method: Southern blot analysis was performed with double digestion, either with EcoRI/EagI or with HindIII/ SacII. The EagI restriction site was studied by sequencing. The fragile X encoded protein (FMRP) was detected in white blood cells by Western blot. The fragile X phenotype was evaluated by specific clinical examinations.
Results: Within one family we found three female carriers of a full mutation and a different degree of methylation of the normal allele that correlated with the levels of FMRP in blood and the fragile X phenotype. Complete methylation at the EagI CpG target (but partially methylated SacII CpG site) was associated with extremely skewed X inactivation (confirmed by analysis of the methylation status at the PGK locus), undetectable FMRP in blood, and a male-like phenotype.
Conclusions: In fully mutated female carriers the methylation status at the EagI restriction site correlates with the levels of FMRP in blood and the fragile X phenotype. Neighboring CpG sequences in the FMR1 promoter can be differentially methylated, which should be taken into consideration for molecular diagnosis.
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Acknowledgements
We would like to express our gratitude to Dr J. López-Barneo for helpful advice during the preparation of this manuscript. We thank Dr J.L. Mandel for the StB12.3 probe. This work was supported by grants from Servicio Andaluz de Salud Exp:80/03 (to E. Pintado) and Exp:22/03 (to M. Lucas). R. Martínez, V. Bonilla-Henao and A. Jiménez were supported by fellowships from Servicio Andaluz de Salud, Seville, Spain.
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Martínez, R., Bonilla-Henao, V., Jiménez, A. et al. Skewed X Inactivation of the Normal Allele in Fully Mutated Female Carriers Determines the Levels of FMRP in Blood and the Fragile X Phenotype. CNS Drugs 9, 157–162 (2005). https://doi.org/10.1007/BF03260084
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DOI: https://doi.org/10.1007/BF03260084