SARS-CoV-2 in Animal Companions: A Serosurvey in Three Regions of Southern Italy.

Several animal species have been found to be susceptible to SARS-CoV-2 infection. The occurrence of infection in dogs and cats living in close contact with owners deserves particular attention from public health authorities in a One Health approach. In this study, we conducted serological screening to identify SARS-CoV-2 exposure in the sera from dogs and cats in three regions of southern Italy sampled during the years 2021 and 2022. We collected 100 serum samples in 2021 (89 from dogs and 11 from cats) and 640 in 2022 (577 from dogs and 63 from cats). Overall, the ELISA positivity rate was found to be 2.7% (20/740), with higher seroprevalence in dogs. Serum neutralization tests confirmed positivity only in two samples collected from dogs, and the assays, performed with serologically distinct SARS-CoV-2 variants, showed variant-specific positivity. This paper shows that monitoring SARS-CoV-2 exposure in animals might be affected by the viral antigenic evolution, which requires continuous updates to the serological tests used. Serological surveys are useful in understanding the true extent of exposure occurring in specific animal populations, not suffering the same limitations as molecular tests, and could help in identifying the infecting virus if tests able to characterize the immune response are used. The use of variant-specific validated serological methods should always be considered in serosurvey studies in order to determine the real impact of emerging variants on animal populations and its implications for veterinary and human health, as well as to identify potential reservoirs of the virus and its evolutionary changes.

Integrative CUT&Tag-RNA-Seq analysis of histone variant macroH2A1-dependent orchestration of human induced pluripotent stem cell reprogramming.

Aim: Human induced pluripotent stem cells (iPSCs) are inefficiently derived from somatic cells by overexpression of defined transcription factors. Overexpression of H2A histone variant macroH2A1.1, but not macroH2A1.2, leads to increased iPSC reprogramming by unclear mechanisms. Materials & methods: Cleavage under targets and tagmentation (CUT&Tag) allows robust epigenomic profiling of a low cell number. We performed an integrative CUT&Tag-RNA-Seq analysis of macroH2A1-dependent orchestration of iPSCs reprogramming using human endothelial cells. Results: We demonstrate wider genome occupancy, predicted transcription factors binding, and gene expression regulated by macroH2A1.1 during reprogramming, compared to macroH2A1.2. MacroH2A1.1, previously associated with neurodegenerative pathologies, specifically activated ectoderm/neural processes. Conclusion: CUT&Tag and RNA-Seq data integration is a powerful tool to investigate the epigenetic mechanisms occurring during cell reprogramming.

Mucosal Microbiota from Colorectal Cancer, Adenoma and Normal Epithelium Reveals the Imprint of Fusobacterium nucleatum in Cancerogenesis.

An increasing amount of evidence suggests the emerging role of the gut microbiota in the development of colorectal cancer (CRC). This study aimed to elucidate the architecture of microbial communities within normal and neoplastic colonic mucosa. METHODS: Microbiota were analyzed by NGS and by an ensemble of metagenomics analysis tools in a total of 69 tissues from 9 patients with synchronous colorectal neoplasia and adenomas (27 specimens: 9 from normal tissues, 9 adenomas, and 9 tumours), 16 patients with only colonic adenomas (32 specimens: 16 from normal tissues and 16 adenomas), and from healthy subjects (10 specimens of normal mucosa). RESULTS: Weak differences were observed in alpha and beta metrics among the synchronous tissues from CRC and controls. Through pairwise differential abundance analyses of sample groups, an increasing trend of Rikenellaceae, Pseudomonas and Fusobacterium, and decreasing trends of Staphylococcus, Actinobacillus and Gemmiger were observed in CRC, while Staphylococcus and Bifidobacterium were decreased in patients with only adenomas. At RT-qPCR analysis, Fusobacterium nucleatum was significantly enriched in all the tissues of subjects with synchronous colorectal neoplasia. CONCLUSION: Our findings provide a comprehensive view of the human mucosa-associated gut microbiota, emphasizing global microbial diversity mostly in synchronous lesions and proving the constant presence of Fusobacterium nucleatum, with its ability to drive carcinogenesis.

Connecting the dots: A practical evaluation of web-tools for describing protein dynamics as networks.

Protein Structure Networks (PSNs) are a well-known mathematical model for estimation and analysis of the three-dimensional protein structure. Investigating the topological architecture of PSNs may help identify the crucial amino acid residues for protein stability and protein-protein interactions, as well as deduce any possible mutational effects. But because proteins go through conformational changes to give rise to essential biological functions, this has to be done dynamically over time. The most effective method to describe protein dynamics is molecular dynamics simulation, with the most popular software programs for manipulating simulations to infer interaction networks being RING, MD-TASK, and NAPS. Here, we compare the computational approaches used by these three tools-all of which are accessible as web servers-to understand the pathogenicity of missense mutations and talk about their potential applications as well as their advantages and disadvantages.

Antimicrobial activity of bovine lactoferrin against Gardnerella species clinical isolates.

Gardnerella species play a key role in the development and recurrence of Bacterial Vaginosis (BV), a common imbalance of the vaginal microbiota. Because of the high rates of BV recurrence reported after treatment with standard of care antibiotics, as well as the emergence of antibiotic-resistant BV, the development of alternative treatment approaches is needed. Bovine lactoferrin, a well studied iron-binding glycoprotein with selective antimicrobial activity, may ameliorate vaginal dysbiosis either alone or in combination with antibiotics. The present study evaluated the antimicrobial resistance/susceptibility profile of seventy-one presumptive G. vaginalis clinical isolates to metronidazole and clindamycin. In addition, the in vitro antimicrobial activity of Metrodora Therapeutics bovine Lactoferrin (MTbLF) against the tested clinical isolates, both alone and in combination with metronidazole and clindamycin, was in depth evaluated using defined-iron culture conditions. All 71 presumptive G. vaginalis clinical isolates exhibited resistance to metronidazole, with MIC values greater than 256 µg/ml. Different susceptibility profiles were detected for clindamycin. In detail, the vast majority of the tested strains (45%), exhibiting MIC lower than 2 µg/ml, were considered sensitive; 18 strains (25%) with MIC higher or equal to 8 µg/ml, were classified as resistant, whereas the remaining 21 (30%) were classified as intermediate. MTbLF was tested in culture medium at different concentrations (32, 16, 8, 4, 2, 1, and 0.5 mg/ml) showing ability to inhibit the growth of the tested presumptive G. vaginalis clinical isolates, including those metronidazole-resistant, in a dose-dependent and not in a strain-dependent manner. MTbLF, at concentrations ranging from 32 to 8 mg/ml, exerted a statistically different antimicrobial activity compared with lower concentrations (4, 2, 1, and 0.5 mg/ml). A synergistic effect between MTbLF (8 and 4 mg/ml) and clindamycin was revealed for all the tested strains. When tested in the absence of other sources of iron, MTbLF did not support the growth of the tested presumptive G. vaginalis clinical isolates. Bovine lactoferrin may be a potential candidate to treat Gardnerella species infection.

Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing.

DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA aberrant methylation events occur simultaneously in a group of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Due to this reason, methylation profiles have been suggested as promising cancer biomarkers. Here, we designed and performed a first step of validation of a novel targeted next generation sequencing (NGS) panel for methylation analysis, which can simultaneously evaluate the methylation levels at CpG sites of multiple cancer-related genes. The OPERA_MET-A methylation panel was designed using the Ion AmpliSeq™ technology to amplify 155 regions with 125-175 bp mean length and covers a total of 1107 CpGs of 18 cancer-related genes. The performance of the panel was assessed by running commercially available fully methylated and unmethylated control human genomic DNA (gDNA) samples and a variable mixture of them. The libraries were run on Ion Torrent platform and the sequencing output was analyzed using the "methylation_analysis" plugin. DNA methylation calls on both Watson (W) and Crick (C) strands and methylated:unmethylated ratio for each CpG site were obtained. Cell lines, fresh frozen and formalin-fixed paraffin-embedded (FFPE) lung cancer tissues were tested. The OPERA_MET-A panel allows to run a minimum of 6 samples/530 chip to reach an observed mean target depth ≥2,500X (W and C strands) and an average number of mapped reads >750,000/sample. The conversion efficiency, determined by spiking-in unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed percentage of global methylation for all CpGs was >95% and <5% for fully methylated and unmethylated gDNA samples, respectively, and the observed results for the variable mixtures were in agreement with what was expected. Methylation-specific NGS analysis represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. Moreover, it offers the opportunity to construct a more robust algorithm for disease prediction in cancer patients having a low quantity of biological material available.

Detection of ALK fusion variants by RNA-based NGS and clinical outcome correlation in NSCLC patients treated with ALK-TKI sequences.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) fusions identify a limited subset of non-small cell lung cancer (NSCLC) patients, whose therapeutic approach have been radically changed in recent years. However, diagnostic procedures and clinical-radiological responses to specific targeted therapies remain heterogeneous and intrinsically resistant or poor responder patients exist. METHODS: A total of 290 patients with advanced NSCLC defined as ALK+ by immunohistochemistry (IHC) and/or fluorescent in situ hybridisation (FISH) test and treated with single or sequential multiple ALK inhibitors (ALKi) from 2011 to 2017 have been retrospectively retrieved from a multicentre Italian cancer network database. In 55 patients with enough leftover tumour tissue, specimens were analysed with both targeted and customised next generation sequencing panels. Identified fusion variants have been correlated with clinical outcomes. RESULTS: Of the 55 patients, 24 received crizotinib as first-line therapy, 1 received ceritinib, while 30 received chemotherapy. Most of the patients (64%) received ALKi in sequence. An ALK fusion variant was identified in 73% of the cases, being V3 variant (E6A20) the most frequent, followed by V1 (E13A20) and more rare ones (e.g. E6A19). In three specimens, four new EML4-ALK fusion breakpoints have been reported. Neither fusion variants nor brain metastases were significantly associated with overall survival (OS), while it was predictably longer in patients receiving a sequence of ALKi. The presence of V1 variant was associated with progression-free survival (PFS) improvement when crizotinib was used (p = 0.0073), while it did not affect cumulative PFS to multiple ALKi. CONCLUSION: Outcomes to sequential ALKi administration were not influenced by fusion variants. Nevertheless, in V1+ patients a prolonged clinical benefit was observed. Fusion variant identification by NGS technology may add relevant information about rare chromosomal events that could be potentially correlated to worse outcomes.

RhythmicDB: A Database of Predicted Multi-Frequency Rhythmic Transcripts.

The physiology and behavior of living organisms are featured by time-related variations driven by molecular clockworks that arose during evolution stochastically and heterogeneously. Over the years, several high-throughput experiments were performed to evaluate time-dependent gene expression in different cell types across several species and experimental conditions. Here, these were retrieved, manually curated, and analyzed by two software packages, BioCycle and MetaCycle, to infer circadian or ultradian transcripts across different species. These transcripts were stored in RhythmicDB and made publically available.

KDM6A missense variants hamper H3 histone demethylation in lung squamous cell carcinoma.

KDM6A is the disease causative gene of type 2 Kabuki Syndrome, a rare multisystem disease; it is also a known cancer driver gene, with multiple somatic mutations found in a few cancer types. In this study, we looked at eleven missense variants in lung squamous cell carcinoma, one of the most common lung cancer subtypes, to see how they affect the KDM6A catalytic mechanisms. We found that they influence the interaction with histone H3 and the exposure of the trimethylated Lys27, which is critical for wild-type physiological function to varying degrees, by altering the conformational transition.

Adherence to Gluten-Free Diet Restores Alpha Diversity in Celiac People but the Microbiome Composition Is Different to Healthy People.

Celiac disease (CD) is an autoimmune disease with the destruction of small intestinal villi, which occurs in genetically predisposed individuals. At the present moment, a gluten-free diet (GFD) is the only way to restore the functionality of gut mucosa. However, there is an open debate on the effects of long-term supplementation through a GFD, because some authors report an unbalance in microbial taxa composition. METHODS: For microbiome analysis, fecal specimens were collected from 46 CD individuals in GFD for at least 2 years and 30 specimens from the healthy controls (HC). Data were analyzed using an ensemble of software packages: QIIME2, Coda-lasso, Clr-lasso, Selbal, PICRUSt2, ALDEx2, dissimilarity-overlap analysis, and dysbiosis detection tests. RESULTS: The adherence to GFD restored the alpha biodiversity of the gut microbiota in celiac people but microbial composition at beta diversity resulted as different to HC. The microbial composition of the CD subjects was decreased in a number of taxa, namely Bifidobacterium longum and several belonging to Lachnospiraceae family, whereas Bacteroides genus was found to be more abundant. Predicted metabolic pathways among the CD bacterial communities revealed an important role in tetrapyrrole biosynthesis. CONCLUSIONS: CD patients in GFD had a non-dysbiotic microbial composition for the crude alpha diversity metrics. We found significant differences in beta diversity, in certain taxon, and pathways between subjects with inactive CD in GFD and controls. Collectively, our data may suggest the development of new GFD products by modulating the gut microbiota through diet, supplements of vitamins, and the addition of specific prebiotics.

Real-time, computer-aided, detection-assisted colonoscopy eliminates differences in adenoma detection rate between trainee and experienced endoscopists.

Background and study aims Adenoma detection rate (ADR) is a well-accepted quality indicator of screening colonoscopy. In recent years, the added value of artificial intelligence (AI) has been demonstrated in terms of ADR and adenoma miss rate (AMR). To date, there are no studies evaluating the impact of AI on the performance of trainee endoscopists (TEs). This study aimed to assess whether AI might eliminate any difference in ADR or AMR between TEs and experienced endoscopists (EEs). Patients and methods We performed a prospective observational study in 45 subjects referred for screening colonoscopy. A same-day tandem examination was carried out for each patient by a TE with the AI assistance and subsequently by an EE unaware of the lesions detected by the TE. Besides ADR and AMR, we also calculated for each subgroup of endoscopists the adenoma per colonoscopy (APC), polyp detection rate (PDR), polyp per colonoscopy (PPC) and polyp miss rate (PMR). Subgroup analyses according to size, morphology, and site were also performed. Results ADR, APC, PDR, and PPC of AI-supported TEs were 38 %, 0.93, 62 %, 1.93, respectively. The corresponding parameters for EEs were 40 %, 1.07, 58 %, 2.22. No significant difference was found for each analysis between the two groups ( P  > 0.05). AMR and PMR for AI-assisted TEs were 12.5 % and 13 %, respectively. Sub-analyses did not show any significant difference ( P  > 0.05) between the two categories of operators. Conclusions In this single-center prospective study, the possible impact of AI on endoscopist quality training was demonstrated. In the future, this could result in better efficacy of screening colonoscopy by reducing the incidence of interval or missed cancers.

Genome-wide DNA methylation profiling and exome sequencing resolved a long-time misdiagnosed case.

The search for aetiology of Mendelian disorders is traditionally based on the observation of clinical phenotypes and molecular screening of associated genes. However, a disease-specific diagnosis can be challenging. In this study we detail how the combinatorial genomic and epigenomic assessment allowed to find the underlying molecular event of a clinical case that remained misdiagnosed for years. The individual was referred as affected by an atypical form of Kabuki syndrome with a variant of uncertain significance in the KMT2D gene. However, significant inconsistencies with this diagnosis emerged upon familial segregation of the variant and after the clinical re-evaluation. Therefore, we applied an epigenomic strategy by studying the DNA methylation profile which resulted not consistent with the Kabuki syndrome episignature or with any other disorder-specific episignature described so far, providing strong evidence that the Kabuki syndrome diagnosis does not apply. This result led us to further investigate for epigenetic machinery diseases by using a multigene panel for chromatinopathies. Since this analysis yielded negative results, we applied a whole exome sequencing and identified a de novo pathogenic variant in the CTNNB1 gene associated to NEDSDV syndrome, a neurodevelopmental disorder characterized by intellectual disability and craniofacial anomalies. Based on molecular results and the updated clinical features, we confirmed the NEDSDV diagnosis. Our findings show that the combination of genomic and epigenomics strategies, along with a deeper analysis of clinical phenotype, may provide a significant improvement in the diagnostic protocols for rare genetic disorders and help resolve long-time misdiagnosed and unsolved case.

Healthy and pro-inflammatory gut ecology plays a crucial role in the digestion and tolerance of a novel Gluten Friendly™ bread in celiac subjects: a randomized, double blind, placebo control in vivo study.

Gluten Friendly™ (GF) is a new gluten achieved through a physicochemical process applied to wheat kernels. The goal of this research was to assess the in vivo effects of Gluten Friendly™ bread on celiac gut mucosa and microbiota. In a double-blind placebo-controlled intervention study, 48 celiac disease (CD) patients were randomized into 3 groups to eat 100 g of bread daily, containing different doses (0; 3 g; 6 g) of GF for 12 weeks. The small-bowel morphology (VH/CrD), intraepithelial densities of CD3+, celiac serology, MUC2, CB1, gut permeability, proinflammatory cytokines, gluten in stools, symptoms, and gut microbial composition were assessed. All 48 CD subjects experienced no symptoms. K-means analysis evidenced celiac subjects clustering around unknown parameters independent of GF dosage: K1 35%; K2 30%; K3 35%. VH/CrD significantly decreased in K1 and K2. VH/CrD did not correlate with IEL increase in K2. 33-mer was not detected in 47% and 73% of patients in both K1 and K2, respectively. VH/CrD and IEL did not change significantly and strongly correlated with the absence of 33-mer in K3. Inflammation and VH/CrD decrease are strongly related with the presence of proinflammatory species at the baseline. A boost in probiotic, butyrate-producing genera, is strongly related with GF tolerance at the end of the trial. Our research suggests that a healthy and proinflammatory ecology could play a crucial role in the digestion and tolerance of the new gluten molecule in celiac subjects. However, GF can be completely digested by gut microbiota of CD subjects and shapes it toward gut homeostasis by boosting healthy butyrate-producing populations. The clinical trial registry number is NCT03137862 (https://clinicaltrials.gov).

Germline Alterations in Patients With IBD-associated Colorectal Cancer.

BACKGROUND: Patients with inflammatory bowel diseases (IBD), both ulcerative colitis (UC) and Crohn's disease (CD), are at risk of developing a colorectal cancer (CRC). No information is available on the contribution of patients' genetic background to CRC occurrence. This study investigates germline alterations in patients with IBD-associated CRC. METHODS: We profiled a panel of 39 genes potentially involved in cancer predisposition and searched for germline variants in IBD patients with CRC or high-grade dysplasia. RESULTS: After clinical exclusion of genetic cancer syndromes, 25 IBD patients (4 CD and 21 UC) with CRC or high-grade dysplasia were studied. After excluding variants with low likelihood of pathogenicity (classes 1 or 2 according the International Agency for Research on Cancer [IARC]), the panel identified pathogenic variants, likely pathogenic, or variants with unknown significance in 18 patients (72%). Six patients (24%) carried pathogenic or likely variants (IARC class 5 or 4). Of the identified variants, 4 encompassed the APC region, 3 the MLH1 gene, and the remaining ones the MSH2, MSH3, monoallelic MUTYH, EPCAM, BRCA1, CHEK2, POLD1, POLE, CDKN2A, and PDGFRA genes. Four patients carried at least 2 variants in different genes. Duration of IBD was significantly shorter in carriers of 4 or 5 IARC variants (7 years; range 0-21; P = .002) and in those with variants with unknown significance (12 years; range 0-22; P = .005) compared with patients without or with only benign variations (23.5 years; range 15-34). CONCLUSIONS: In silico analysis and sequence-based testing of germline DNA from IBD patients with CRC or high-grade dysplasia detected 24% of variants positioned in pathogenic classes. In patients with type 3, 4, and 5 variants, the onset of high-grade dysplasia or CRC was significantly earlier than in patients with benign or unidentified variants. The screening for these genes could identify IBD patients requiring a more intensive endoscopic surveillance for earlier detection of dysplastic changes.

Microbiome Analysis of Mucosal Ileoanal Pouch in Ulcerative Colitis Patients Revealed Impairment of the Pouches Immunometabolites.

The pathogenesis of ulcerative colitis (UC) is unknown, although genetic loci and altered gut microbiota have been implicated. Up to a third of patients with moderate to severe UC require proctocolectomy with ileal pouch ano-anastomosis (IPAA). We aimed to explore the mucosal microbiota of UC patients who underwent IPAA. METHODS: For microbiome analysis, mucosal specimens were collected from 34 IPAA individuals. Endoscopic and histological examinations of IPAA were normal in 21 cases, while pouchitis was in 13 patients. 19 specimens from the healthy control (10 from colonic and 9 from ileum) were also analyzed. Data were analyzed using an ensemble of software packages: QIIME2, coda-lasso, clr-lasso, PICRUSt2, and ALDEx2. RESULTS: IPAA specimens had significantly lower bacterial diversity as compared to normal. The microbial composition of the normal pouch was also decreased also when compared to pouchitis. Faecalibacterium prausnitzii, Gemmiger formicilis, Blautia obeum, Ruminococcus torques, Dorea formicigenerans, and an unknown species from Roseburia were the most uncommon in pouch/pouchitis, while an unknown species from Enterobacteriaceae was over-represented. Propionibacterium acnes and Enterobacteriaceae were the species most abundant in the pouchitis and in the normal pouch, respectively. Predicted metabolic pathways among the IPAA bacterial communities revealed an important role of immunometabolites such as SCFA, butyrate, and amino acids. CONCLUSIONS: Our findings showed specific bacterial signature hallmarks of dysbiosis and could represent bacterial biomarkers in IPAA patients useful to develop novel treatments in the future by modulating the gut microbiota through the administration of probiotic immunometabolites-producing bacterial strains and the addition of specific prebiotics and the faecal microbiota transplantation.

Novel STAG1 Frameshift Mutation in a Patient Affected by a Syndromic Form of Neurodevelopmental Disorder.

The cohesin complex is a large evolutionary conserved functional unit which plays an essential role in DNA repair and replication, chromosome segregation and gene expression. It consists of four core proteins, SMC1A, SMC3, RAD21, and STAG1/2, and by proteins regulating the interaction between the complex and the chromosomes. Mutations in the genes coding for these proteins have been demonstrated to cause multisystem developmental disorders known as "cohesinopathies". The most frequent and well recognized among these distinctive clinical conditions are the Cornelia de Lange syndrome (CdLS, OMIM 122470) and Roberts syndrome (OMIM 268300). STAG1 belongs to the STAG subunit of the core cohesin complex, along with five other subunits. Pathogenic variants in STAG1 gene have recently been reported to cause an emerging syndromic form of neurodevelopmental disorder that is to date poorly characterized. Here, we describe a 5 year old female patient with neurodevelopmental delay, mild intellectual disability, dysmorphic features and congenital anomalies, in which next generation sequencing analysis allowed us to identify a novel pathogenic variation c.2769_2770del p.(Ile924Serfs*8) in STAG1 gene, which result to be de novo. The variant has never been reported before in medical literature and is absent in public databases. Thus, it is useful to expand the molecular spectrum of clinically relevant alterations of STAG1 and their phenotypic consequences.

A comparative benchmark of classic DNA motif discovery tools on synthetic data.

Hundreds of human proteins were found to establish transient interactions with rather degenerated consensus DNA sequences or motifs. Identifying these motifs and the genomic sites where interactions occur represent one of the most challenging research goals in modern molecular biology and bioinformatics. The last twenty years witnessed an explosion of computational tools designed to perform this task, whose performance has been last compared fifteen years ago. Here, we survey sixteen of them, benchmark their ability to identify known motifs nested in twenty-nine simulated sequence datasets, and finally report their strengths, weaknesses, and complementarity.

Phenotypic Variability of a Pathogenic PKP2 Mutation in an Italian Family Affected by Arrhythmogenic Cardiomyopathy and Juvenile Sudden Death: Considerations From Molecular Autopsy to Sport Restriction.

Background: Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder with an estimated prevalence between 1:2,000 and 1:5,000 and is characterized by the fibrofatty replacement of cardiomyocytes that predisposes to malignant arrhythmias, heart failure, and sudden cardiac death. The diagnosis is based on the 2010 Task Force Criteria including family history, electrocardiographic traits and arrhythmogenic pattern, specific gene mutations, and structural and/or histological abnormalities. Most ACMs display an autosomal dominant mode of inheritance often with incomplete penetrance and variable expressivity. Genetic screening of patients with ACM identifies pathogenic or likely pathogenic variants, prevalently in genes encoding the cardiac desmosome (PKP2, DSP, DSC2, DSG2, and JUP) or less frequently in non-desmosomal genes (CTNNA3, PLN, TMEM43, RYR2, SCN5A, CDH2, and DES). Methods: In the present study, we performed molecular autopsy in a boy who died suddenly during physical exertion. In addition to post-mortem examination, a DNA sample was analyzed with next-generation sequencing (NGS). Results: The genetic analysis revealed the presence of pathogenic heterozygous c.314del (p.Pro105Leufs*7) frameshift variant in the PKP2 gene. Cascade screening of family members allowed us to identify 12 mutation carriers and to intervene on subjects at risk, many of whom were athletes. Conclusions: Molecular autopsy can establish cardiogenetic diagnosis and allow appropriate preventative measures in high-risk relatives.

Whole Exome Sequencing Reveals a Novel AUTS2 In-Frame Deletion in a Boy with Global Developmental Delay, Absent Speech, Dysmorphic Features, and Cerebral Anomalies.

Neurodevelopmental disorders (NDDs) are a group of highly prevalent, clinically and genetically heterogeneous pediatric disorders comprising, according to the Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-V), intellectual disability, developmental delay, autism spectrum disorders, and other neurological and cognitive disorders manifesting in the developmental age. To date, more than 1000 genes have been implicated in the etiopathogenesis of NNDs. Among them, AUTS2 (OMIM # 607270) encodes a protein involved in neural migration and neuritogenesis, and causes NNDs with different molecular mechanisms including copy number variations, single or multiple exonic deletion and single nucleotide variants. We describes a 9-year-old boy with global developmental delay, absent speech, minor craniofacial anomalies, hypoplasia of the cerebellar vermis and thinning of the corpus callosum, resulted carrier of the de novo AUTS2 c.1603_1626del deletion at whole exome sequencing (WES) predicted to cause the loss of eight amino acids [p.(His535_Thr542del)]. Notably, our patient is the first reported so far in medical literature carrying an in-frame deletion and the first in which absent language, hypoplasia of the cerebellar vermis and thinning of the corpus callosum has been observed thus useful to expand the molecular spectrum of AUTS2 pathogenic variants and to broaden our knowledge on the clinical phenotype associated.

Expanding the phenotype associated to KMT2A variants: overlapping clinical signs between Wiedemann-Steiner and Rubinstein-Taybi syndromes.

Lysine-specific methyltransferase 2A (KMT2A) is responsible for methylation of histone H3 (K4H3me) and contributes to chromatin remodeling, acting as "writer" of the epigenetic machinery. Mutations in KMT2A were first reported in Wiedemann-Steiner syndrome (WDSTS). More recently, KMT2A variants have been described in probands with a specific clinical diagnosis comprised in the so-called chromatinopathies. Such conditions, including WDSTS, are a group of overlapping disorders caused by mutations in genes coding for the epigenetic machinery. Among them, Rubinstein-Taybi syndrome (RSTS) is mainly caused by heterozygous pathogenic variants in CREBBP or EP300. In this work, we used next generation sequencing (either by custom-made panel or by whole exome) to identify alternative causative genes in individuals with a RSTS-like phenotype negative to CREBBP and EP300 mutational screening. In six patients we identified different novel unreported variants in KMT2A gene. The identified variants are de novo in at least four out of six tested individuals and all of them display some typical RSTS phenotypic features but also WDSTS specific signs. This study reinforces the concept that germline variants affecting the epigenetic machinery lead to a shared molecular effect (alteration of the chromatin state) determining superimposable clinical conditions.