Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells

Background: Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5\u2032 UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously demonstrated that 5-aza-2-deoxycytidine (5-azadC) treatment of FXS lymphoblastoid cell lines reactivates the FMR1 gene, concomitant with CpG sites demethylation, increased acetylation of histones H3 and H4 and methylation of lysine 4 on histone 3. Results: In order to check the specificity of the 5-azadC-induced DNA demethylation, now we performed bisulphite sequencing of the entire methylation boundary upstream the FMR1 promoter region, which is preserved in control wild-type cells. We did not observe any modification of the methylation boundary after treatment. Furthermore, methylation analysis by MS-MLPA of PWS/AS and BWS/SRS loci demonstrated that 5-azadC treatment has no demethylating effect on these regions. Genome-wide methylation analysis through Infinium 450K (Illumina) showed no significant enrichment of specific GO terms in differentially methylated regions after 5-azadC treatment. We also observed that reactivation of FMR1 transcription lasts up to a month after a 7-day treatment and that maximum levels of transcription are reached at 10-15 days after last administration of 5-azadC. Conclusions: Taken together, these data demonstrate that the demethylating effect of 5-azadC on genomic DNA is not random, but rather restricted to specific regions, if not exclusively to the FMR1 promoter. Moreover, we showed that 5-azadC has a long-lasting reactivating effect on the mutant FMR1 gene

Genetic counseling during COVID‐19 pandemic: Tuscany experience

Abstract Background COVID‐19 outbreak prompted health centres to reorganize their clinical and surgical activity. In this paper, we show how medical genetics department's activity, in our tertiary pediatric hospital, has changed due to pandemic. Methods We stratified all our scheduled visits, from March 9th through April 30th, and assessed case‐by‐case which genetic consultations should be maintained as face‐to‐face visit, or postponed/switched to telemedicine. Results Out of 288 scheduled appointments, 60 were prenatal consultations and 228 were postnatal visits. We performed most of prenatal consultations as face‐to‐face visits, as women would have been present in the hospital to perform other procedures in addition to our consult. As for postnatal care, we suspended all outpatient first visits and opted for telemedicine for selected follow‐up consultations: interestingly, 75% of our patients’ parents revealed that they would have cancelled the appointment themselves for the fear to contract an infection. Conclusions Spread of COVID‐19 in Italy forced us to change our working habits. Given the necessity to optimize healthcare resources and minimize the risk of in‐hospital infections, we experienced the benefits of telegenetics. Current pandemic made us familiar with telemedicine, laying the foundations for its application to deal with the increasing number of requests in clinical genetics

An autosomal recessive DNASE1L3-related autoimmune disease with unusual clinical presentation mimicking systemic lupus erythematosus

We describe the third family in the world, after Arabian and Turkish ones, displaying an autosomal recessive autoimmune disease (AID), mimicking systemic lupus erythematosus (SLE), with unusual manifestations due to a homozygous frame-shift variant in DNASE1L3. SLE is a complex AID characterized by multiple organ involvement. Genetic risk variants identified account for only 15% of SLE heritability. Rare Mendelian forms have been reported, including DNASE1L3-related SLE. Through specific genetic tests we identified a homozygous 2 bp-deletion c.289_290delAC (NM_004944.2) in DNASE1L3, predicting frameshift and premature truncation (p.Thr97Ilefs*2). The same mutation was previously reported in three sisters, born from consanguineous parents and affected with hypocomplementemic urticarial vasculitis syndrome (HUVS). As approximately 50% of individuals affected with HUVS develop SLE, it is still unclear whether it is a SLE sub-phenotype or a separate condition. Lupus (2016) 0, 1\u20135

ulcera peptica

<div><p>Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the <i>FMR1</i> gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (<i>FMR1</i>-<i>AS1</i>), starting from both promoter and intron 2 of the <i>FMR1</i> gene, was demonstrated in transcriptionally active alleles, but not in silent FXS alleles. Moreover, a DNA methylation boundary, which is lost in FXS, was recently identified upstream of the <i>FMR1</i> gene. Several nuclear proteins bind to this region, like the insulator protein CTCF. Here we demonstrate for the first time that rare unmethylated full mutation (UFM) alleles present the same boundary described in wild type (WT) alleles and that CTCF binds to this region, as well as to the <i>FMR1</i> gene promoter, exon 1 and intron 2 binding sites. Contrariwise, DNA methylation prevents CTCF binding to FXS alleles. Drug-induced CpGs demethylation does not restore this binding. <i>CTCF</i> knock-down experiments clearly established that CTCF does not act as insulator at the active <i>FMR1</i> locus, despite the presence of a CGG expansion. <i>CTCF</i> depletion induces heterochromatinic histone configuration of the <i>FMR1</i> locus and results in reduction of <i>FMR1</i> transcription, which however is not accompanied by spreading of DNA methylation towards the <i>FMR1</i> promoter. <i>CTCF</i> depletion is also associated with <i>FMR1-AS1</i> mRNA reduction. Antisense RNA, like sense transcript, is upregulated in UFM and absent in FXS cells and its splicing is correlated to that of the <i>FMR1</i>-mRNA. We conclude that CTCF has a complex role in regulating <i>FMR1</i> expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis.</p></div

<i>CTCF</i> knock-down in WT and UFM fibroblasts.

<p>(<b>A</b>) Western blotting analysis of CTCF, visualized with ECL kit. Protein extracts from untreated (UT) and siRNA-treated WT and UFM fibroblasts were probed with an anti-CTCF rabbit polyclonal antibody (top panel) and one against GAPDH (bottom panel). After <i>CTCF</i> depletion a major reduction of the corresponding protein is visible. (<b>B</b>) Relative quantification through RT-PCR of <i>CTCF</i> and <i>FMR1</i> sense and antisense transcripts in those knock-down experiments in which <i>CTCF</i> depletion is followed by <i>FMR1</i> transcript reduction both in WT (upper panel) and UFM (bottom panel) fibroblasts. <i>FMR1</i> sense transcript is reduced to around 50% in both cell lines with a consistent (about 80%) reduction of <i>CTCF</i> mRNA levels. Depletion of <i>FMR1</i>-<i>AS1</i> (80 and 60% in WT and UFM cells, respectively) is also observed and directly correlates with <i>CTCF</i> reduction in both cell lines. The two cell lines were also transfected with a scramble siRNA (scRNA) without any modifications of <i>CTCF</i> transcript levels. Histograms represent mean and standard deviation of 10 independent knock-down experiments for UFM and WT.</p

Results obtained from SVM prediction system.

<p>The first column lists the possible combination of CTCF binding sites, as already reported (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003601#pgen-1003601-g002" target="_blank">Figure 2</a>). Columns 2 and 3 report the prediction (L = predicted loop, NL = predicted non-loop) and the probability of WT allele for each putative loop, while columns 4 and 5 report results of expanded CGG allele (>200 repeats). Probability is an accuracy index of prediction, higher is its value more confident is the prediction. In bold are reported the more probable loops.</p

<i>CTCF</i> overexpression in WT, UFM and FXS fibroblasts.

<p><i>CTCF</i> overexpression in WT, UFM and FXS cell lines after transfection with a commercial vector containing the open reading frame of variant 1 of <i>CTCF</i>. Quantitative RT-PCR showed a strong increase of <i>CTCF</i> mRNA after 48 and 120 hours from transfection (<b>A</b>), while levels of <i>FMR1</i> transcription remained substantially unchanged (<b>B</b>). The levels of <i>CTCF</i> transcription in the untreated cells were arbitrarily set at 1 as well as those of <i>FMR1</i> transcript in WT and UFM fibroblasts, while those of <i>FMR1</i>-mRNA in FXS cells were set at 0. Bars indicate standard deviation.</p