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.

MED13L haploinsufficiency has recently been described as responsible for syndromic intellectual disability. We planned a search for causative gene variants in seven subjects with intellectual disability and overlapping dysmorphic facial features such as bulbous nasal tip, short mouth and straight eyebrows. We found two de novo frameshift variants in MED13L, consisting in single-nucleotide deletion (c.3765delC) and duplication (c.607dupT). A de novo nonsense variant (c.4420A>T) in MED13L was detected in a further subject in the course of routine whole-exome sequencing. By analyzing the clinical data of our patients along with those recently described in the literature, we confirm that there is a common, recognizable phenotype associated with MED13L haploinsufficiency, which includes intellectual disability and a distinctive facial appearance. Congenital heart diseases are found in some subjects with various degree of severity. Our observation of cleft palate, ataxia, epilepsy and childhood leukemia observed in single cases broadens the known clinical spectrum. Haploinsufficiency for MED13L should be considered in the differential diagnosis of the 1p36 microdeletion syndrome, due to overlapping dysmorphic facial features in some patients. The introduction of massive parallel-sequencing techniques into clinical practice is expected to allow for detection of other causative point variants in MED13L. Analysis of genomic data in connection with deep clinical evaluation of patients could elucidate genetic heterogeneity of the MED13L haploinsufficiency phenotype.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the FMR1 gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (FMR1-AS1), starting from both promoter and intron 2 of the FMR1 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 FMR1 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 FMR1 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. CTCF knock-down experiments clearly established that CTCF does not act as insulator at the active FMR1 locus, despite the presence of a CGG expansion. CTCF depletion induces heterochromatinic histone configuration of the FMR1 locus and results in reduction of FMR1 transcription, which however is not accompanied by spreading of DNA methylation towards the FMR1 promoter. CTCF depletion is also associated with FMR1-AS1 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 FMR1-mRNA. We conclude that CTCF has a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis. Fragile X syndrome is the most common cause of inherited intellectual disability, accounting for about 1∶3000 males and 1∶4000 females. It is caused by a dynamic mutation of FMR1, a gene mapping on the X chromosome and containing a CGG repeat in its promoter region. Expansion of this unstable sequence beyond 200 repeats (full mutation) is followed by DNA methylation and histone changes, leading to the transcriptional inactivation of FMR1 and to the lack of the FMRP protein. Recently, an antisense transcript (FMR1-AS1) spanning the CGG repeats and a region of transition of DNA methylation (boundary) located upstream of the CGG repeats have been identified in transcriptional active FMR1 alleles. Several nuclear proteins bound to the methylation boundary have been described, such as the zinc-finger protein CTCF, the first known insulator in mammals. This protein is an important transcriptional regulator of genes harboring trinucleotide repeats and it is mostly active in chromatin organization. For the first time, we have investigated the role of CTCF protein in the transcriptional regulation of the FMR1 gene. Our results define a complex role for CTCF acting through chromatin organization of the FMR1 locus.

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.

Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5′ 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. 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. 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.