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Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3 . Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance. Most cases of craniofacial microsomia are sporadic but familial cases have been reported. Here the authors report that variants in FOXI3 can cause a small fraction of cases with different modes of inheritance including autosomal dominant with reduced penetrance.

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. In this paper, we show how medical genetics department's activity, in our tertiary pediatric hospital, has changed due to COVID‐19 pandemic. 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.

This is the first case of NBAS disease detected by NBS for primary immunodeficiency. NBS with KRECs is revealing unknown potentialities detecting conditions that benefit from early recognition like NBAS deficiency. Immune phenotyping should be mandatory in patients with NBAS deficiency since they can exhibit severe immunodeficiency with hypogammaglobulinemia as the most frequent finding. Fever during infections is a known trigger of acute liver failure in this syndrome, so immune dysfunction, should never go unnoticed in NBAS deficiency in order to start adequate therapy and prophylaxis.

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.

Abstract Disclosure: G. Del Medico: None. E. Andreucci: None. S. Bargiacchi: None. G. Gori: None. G. Mancano: None. G. Traficante: None. F. Girolami: None. S. Favilli: None. A. Peron: None. I. Olivotto: None. M. Tartaglia: None. S. Stagi: None. Background: Noonan syndrome (NS) is a congenital genetic condition with an estimated incidence of 1 in 1,000-2,500 live births. Typical clinical features include congenital heart defects, short stature, distinctive facial characteristics, chest anomalies, and varying degrees of cognitive impairment. NS is caused by pathogenic variants in genes involved in the RAS/MAPK signaling cascade, resulting in significant clinical and genetic variability. Aims: To analyze the prevalence of specific genetic mutations and their correlation to clinical features in NS. Methods: This retrospective observational study included 111 individuals with genetically confirmed NS. Data collected comprised genotype, cardiac abnormalities, and anthropometric measurements (birth weight, birth length, height, and weight at the latest clinical visit). Standard deviation (SD) scores were determined using Italian population growth references. Results: Complete data were available for 60 NS patients (38% male). The most prevalent mutations were identified in PTPN11 (60%) and SOS1 (10%), with additional variants detected in LZTR1, RAF1, RIT1, CBL, KRAS, SOS2, BRAF, and MAP2K1. Cardiovascular abnormalities were present in 62% of the cohort, with PTPN11 and RIT1 mutations predominantly linked to pulmonary valve stenosis, while RAF1 mutations were associated with hypertrophic cardiomyopathy. Less frequent heart defects, such as atrial and ventricular septal defects and abnormalities of the mitral or aortic valves, showed no clear genotype correlation. Small for gestational age (SGA) was identified in 5% of individuals, all carrying PTPN11 mutations. At an average age of 9.8 ± 5.9 years, mean height and weight were -1.8 ± 1.18 SD and -1.5 ± 1.40 SD, respectively. Short stature was present in 42% of patients, with an average height of -2.84 SD. Growth hormone treatment was administered to 37% of patients, the significant majority of whom carried PTPN11 mutations (74%, p<0.05). Among those born SGA, two-thirds developed short stature. Interestingly, individuals with CBL, SOS2, or MAP2K1 mutations did not exhibit short stature. In 28% of the cohort, both short stature and heart defects coexisted, though no significant genotype association was observed for this combination. Conclusion: This study underscores the high frequency of cardiac defects and short stature in NS, with distinct genotype-phenotype correlation for cardiovascular abnormalities. While being born SGA was relatively rare, it was strongly associated with the PTPN11 genotype. Conversely, genotype-growth correlations were less evident, though some mutations appeared to reduce the likelihood of short stature. These findings highlight the genetic and clinical variability of NS and emphasize the necessity for larger studies to enhance our understanding of genotype-phenotype correlations. Presentation: Saturday, July 12, 2025

Fragile X syndrome is the most common heritable form of intellectual disability and is caused by the expansion over 200 repeats and subsequent methylation of the CGG triplets at the 5' UTR of the FMR1 gene, leading to its silencing. The epigenetic and molecular mechanisms responsible for FMR1 gene silencing are not fully clarified. To identify structure-specific proteins that could recruit components of the silencing machinery we investigated the role of CGGBP1 in FMR1 gene transcription. CGGBP1 is a highly conserved protein that binds specifically to unmethylated CGG tracts. Its role on FMR1 transcription is yet to be defined. Sequencing analysis and expression studies through quantitative PCR of CGGBP1 were performed in cell lines with different allele expansions: wild type, premutation, methylated full mutation and unmethylated full mutation, demonstrating no differences between them. ChIP assays clearly demonstrated that CGGBP1 binds to unmethylated CGG triplets of the FMR1 gene, but not to methylated CGGs. We also observed that CGGBP1 binding to the FMR1 locus was restored after pharmacological demethylation, with 5-azadC, of alleles, carriers of methylated full mutation, suggesting a possible role for CGGBP1 in FMR1 expression. CGGBP1 silencing with shRNAs (reaching ~98% of CGGBP1-mRNA depletion) did not affect FMR1 transcription and CGG expansion stability in expanded alleles. Although the strong binding to the CGG tract could suggest a relevant role of CGGBP1 on FMR1 gene expression, our results demonstrate that CGGBP1 has no direct effect on FMR1 transcription and CGG repeat stability.

Background Targeted resequencing has become the most used and cost-effective approach for identifying causative mutations of Mendelian diseases both for diagnostics and research purposes. Due to very rapid technological progress, NGS laboratories are expanding their capabilities to address the increasing number of analyses. Several open source tools are available to build a generic variant calling pipeline, but a tool able to simultaneously execute multiple analyses, organize, and categorize the samples is still missing. Results Here we describe VarGenius, a Linux based command line software able to execute customizable pipelines for the analysis of multiple targeted resequencing data using parallel computing. VarGenius provides a database to store the output of the analysis (calling quality statistics, variant annotations, internal allelic variant frequencies) and sample information (personal data, genotypes, phenotypes). VarGenius can also perform the “joint analysis” of hundreds of samples with a single command, drastically reducing the time for the configuration and execution of the analysis. VarGenius executes the standard pipeline of the Genome Analysis Tool-Kit (GATK) best practices (GBP) for germinal variant calling, annotates the variants using Annovar, and generates a user-friendly output displaying the results through a web page. VarGenius has been tested on a parallel computing cluster with 52 machines with 120GB of RAM each. Under this configuration, a 50 M whole exome sequencing (WES) analysis for a family was executed in about 7 h (trio or quartet); a joint analysis of 30 WES in about 24 h and the parallel analysis of 34 single samples from a 1 M panel in about 2 h. Conclusions We developed VarGenius, a “master” tool that faces the increasing demand of heterogeneous NGS analyses and allows maximum flexibility for downstream analyses. It paves the way to a different kind of analysis, centered on cohorts rather than on singleton. Patient and variant information are stored into the database and any output file can be accessed programmatically. VarGenius can be used for routine analyses by biomedical researchers with basic Linux skills providing additional flexibility for computational biologists to develop their own algorithms for the comparison and analysis of data. The software is freely available at: https://github.com/frankMusacchia/VarGenius

Background 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. 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.

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.