Raquel Chillón Martínez

Skewed X Inactivation of the Normal Allele in Fully Mutated Female Carriers Determines the Levels of FMRP in Blood and the Fragile X Phenotype

AbstractBackground: 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.

Different signaling pathways inhibit DNA methylation activity and up‐regulate IFN‐γ in human lymphocytes

DNA methylation is recognized increasingly for its prominent role in controlling diverse immune processes. In this study, we show that in Jurkat T cells and fresh peripheral lymphocytes, short‐time incubation with protein kinase C activators or phosphatase inhibitors down‐regulate DNA methylation activity in a dose‐dependent manner. This inhibition correlates with the induction of the interferon‐γ (IFN‐γ) gene, which contains several CG sequences in its promoter. The expression of mRNA and protein of the different DNA methyltransferases did not decrease after the treatment. In addition, sulfydryl reagents have a strong inhibitory effect on DNA methylation activity and also induce IFN‐γ gene expression, thus suggesting a link between both effects.

Spanish Version of the Broome Pelvic Muscle Self-Efficacy Scale: Validity and Reliability

Background Self-efficacy appears to be an important predictor of functional recovery for women with urinary incontinence, but no specific Spanish-language questionnaires for measuring pelvic-floor exercise self-efficacy exist. Objective The aim of this study was to design a valid and reliable Spanish version of the Broome Pelvic Muscle Self-Efficacy Scale to measure self-efficacy, as perceived by women with urinary incontinence, in performing pelvic-floor exercises. Design This was an observational validation study. Methods Translation-back translation was used to design the survey, and then the survey was validated with a sample of 119 women who were incontinent and had undergone a pelvic-floor exercise training program. The reliability and construct validity of the questionnaire were assessed. Descriptive statistics were used to score the questionnaire. Internal consistency was evaluated with the Cronbach alpha coefficient and the Pearson correlation coefficient. Exploratory factor analysis with both the principal components extraction method and the varimax rotation method was used to assess construct validity. Results The reliability coefficient (Cronbach alpha=.91) and the correlations among items were high. The factor analysis revealed that 6 main factors accounted for 75.8% of the variance. Limitations Conclusions regarding the validity of the questionnaire should be drawn with caution because of the inability to assess criterion-related validity. Conclusions The Spanish version of the Broome questionnaire for self-efficacy appears to be useful as a measuring tool for a psychometrically accurate, clinically relevant estimation of womens self-efficacy in performing pelvic-floor exercises.

May anomalous X chromosome methylation be responsible for the spontaneous abortion of a male foetus

Pregnancy loss is an important reproductive problem which appears to be highly associated with genetic factors. A spontaneous abortion occurred before prenatal diagnosis could be performed, in a woman who carried a fragile X full mutation. DNA extracted from formalin-fixed paraffin-embedded chorionic villi preparations indicated that it was a male embryo with an apparently methylated X chromosome. The previous analysis of the family showed that her daughter, who also carried a full mutation, exhibited an extremely skewed X inactivation of the normal allele (100%) and a severe fragile X phenotype. Thus, we speculate that the aberrant pattern of X chromosome methylation in this family may provoke the spontaneous miscarriage of this pregnancy that could be explained by at least partial inactivation of the unique X chromosome in a male foetus. Spontaneous abortion occurs quite frequently in humans, and recurrent pregnancy loss is a significant problem in women’s health (Christiansen 2006). Many cases of spontaneous abortion defy diagnosis and genetic factors have been proposed as a major contribution (Lanasa and Hogge 2000; Sierra and Stephenson 2006). The X chromosome inactivation (XCI) is the process in which one of the two X chromosomes present in each cell of female mammals is inactivated during early embryogenesis, to achieve dosage compensation with males (Avner and Heard 2001; Heard 2004). Initial steps of XCI involve a ‘counting process’, which senses the X chromosome/autosome ratio that restricts XCI to female embryos and, thereafter, the choice of which chromosome is inactivated (Morey et al. 2004).