Characterization of a novel HDAC2 pathogenetic variant: a missing puzzle piece for chromatinopathies.

Histone deacetylases (HDACs) are enzymes pivotal for histone modification (i.e. acetylation marks removal), chromatin accessibility and gene expression regulation. Class I HDACs (including HDAC1, 2, 3, 8) are ubiquitously expressed and they often participate in multi-molecular protein complexes. To date, three neurodevelopmental disorders caused by mutations in genes encoding for HDACs (HDAC4, HDAC6 and HDAC8) and thus belonging to the group of chromatinopathies, have been described. We performed whole exome sequencing (WES) for a patient (#249) clinically diagnosed with the chromatinopathy Rubinstein-Taybi syndrome (RSTS) but negative for mutations in RSTS genes, identifying a de novo frameshift variant in HDAC2 gene. We then investigated its molecular effects in lymphoblastoid cell lines (LCLs) derived from the patient compared to LCLs from healthy donors (HD). As the variant was predicted to be likely pathogenetic and to affect the sequence of nuclear localization signal, we performed immunocytochemistry and lysates fractionation, observing a nuclear mis-localization of HDAC2 compared to HD LCLs. In addition, HDAC2 total protein abundance resulted altered in patient, and we found that newly identified variant in HDAC2 affects also acetylation levels, with significant difference in acetylation pattern among patient #249, HD and RSTS cells and in expression of a known molecular target. Remarkably, RNA-seq performed on #249, HD and RSTS cells shows differentially expressed genes (DEGs) common to #249 and RSTS. Interestingly, our reported patient was clinically diagnosed with RSTS, a chromatinopathy which known causative genes encode for enzymes antagonizing HDACs. These results support the role of HDAC2 as causative gene for chromatinopathies, strengthening the genotype-phenotype correlations in this relevant group of disorders.

Prenatal origin of NUTM1 gene rearrangement in infant B-cell precursor acute lymphoblastic leukaemia.

Rearrangement of NUTM1 gene (NUTM1r) is one of the most frequent aberrations occurring in infants (younger than 1 year at diagnosis) with B-cell precursor Acute Lymphoblastic Leukaemia (BCP-ALL). In this study we had the unique opportunity to analyze the umbilical cord blood (UCB) sample from one infant patient with NUTM1r BCP-ALL. Herein we reported for the first time that NUTM1r infant ALL arise prenatally, as both the patient-specific CUX1::NUTM1 fusion gene, as well as two IG/TR leukaemic markers were already present and detectable in the patient's UCB at birth. Our results clearly demonstrate the prenatal origin of NUTM1r infant BCP-ALL.

Biallelic PI4KA Mutations Disrupt B-Cell Metabolism and Cause B-Cell Lymphopenia and Hypogammaglobulinemia.

PURPOSE: PI4KA-related disorder is a highly clinically variable condition characterized by neurological (limb spasticity, developmental delay, intellectual disability, seizures, ataxia, nystagmus) and gastrointestinal (inflammatory bowel disease and multiple intestinal atresia) manifestations. Although features consistent with immunodeficiency (autoimmunity/autoinflammation and recurrent infections) have been reported in a subset of patients, the burden of B-cell deficiency and hypogammaglobulinemia has not been extensively investigated. We sought to describe the clinical presentation and manifestations of patients with PI4KA-related disorder and to investigate the metabolic consequences of biallelic PI4KA variants in B cells. METHODS: Clinical data from patients with PI4KA variants were obtained. Multi-omics analyses combining transcriptome, proteome, lipidome and metabolome analyses in conjunction with functional assays were performed in EBV-transformed B cells. RESULTS: Clinical and laboratory data of 13 patients were collected. Recurrent infections (7/13), autoimmune/autoinflammatory manifestations (5/13), B-cell deficiency (8/13) and hypogammaglobulinemia (8/13) were frequently observed. Patients' B cells frequently showed increased transitional and decreased switched memory B-cell subsets. Pathway analyses based on differentially expressed transcripts and proteins confirmed the central role of PI4KA in B cell differentiation with altered B-cell receptor (BCR) complex and signalling. By altering lipids production and tricarboxylic acid cycle regulation, and causing increased endoplasmic reticulum stress, biallelic PI4KA mutations disrupt B cell metabolism inducing mitochondrial dysfunction. As a result, B cells show hyperactive PI3K/mTOR pathway, increased autophagy and deranged cytoskeleton organization. CONCLUSION: By altering lipid metabolism and TCA cycle, impairing mitochondrial activity, hyperactivating mTOR pathway and increasing autophagy, PI4KA-related disorder causes a syndromic inborn error of immunity presenting with B-cell deficiency and hypogammaglobulinemia.

Extracellular vesicles from II trimester human amniotic fluid as paracrine conveyors counteracting oxidative stress.

BACKGROUND: We previously demonstrated that the human amniotic fluid (hAF) from II trimester of gestation is a feasible source of stromal progenitors (human amniotic fluid stem cells, hAFSC), with significant paracrine potential for regenerative medicine. Extracellular vesicles (EVs) separated and concentrated from hAFSC secretome can deliver pro-survival, proliferative, anti-fibrotic and cardioprotective effects in preclinical models of skeletal and cardiac muscle injury. While hAFSC-EVs isolation can be significantly influenced by in vitro cell culture, here we profiled EVs directly concentrated from hAF as an alternative option and investigated their paracrine potential against oxidative stress. METHODS: II trimester hAF samples were obtained as leftover material from prenatal diagnostic amniocentesis following written informed consent. EVs were separated by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were assessed by nanoparticle tracking analysis, transmission electron microscopy, Western Blot, and flow cytometry; their metabolic activity was evaluated by oximetric and luminometric analyses and their cargo profiled by proteomics and RNA sequencing. hAF-EV paracrine potential was tested in preclinical in vitro models of oxidative stress and dysfunction on murine C2C12 cells and on 3D human cardiac microtissue. RESULTS: Our protocol resulted in a yield of 6.31 ± 0.98 × 109 EVs particles per hAF milliliter showing round cup-shaped morphology and 209.63 ± 6.10 nm average size, with relevant expression of CD81, CD63 and CD9 tetraspanin markers. hAF-EVs were enriched in CD133/1, CD326, CD24, CD29, and SSEA4 and able to produce ATP by oxygen consumption. While oxidative stress significantly reduced C2C12 survival, hAF-EV priming resulted in significant rescue of cell viability, with notable recovery of ATP synthesis and concomitant reduction of cell damage and lipid peroxidation activity. 3D human cardiac microtissues treated with hAF-EVs and experiencing H2O2 stress and TGFß stimulation showed improved survival with a remarkable decrease in the onset of fibrosis. CONCLUSIONS: Our results suggest that leftover samples of II trimester human amniotic fluid can represent a feasible source of EVs to counteract oxidative damage on target cells, thus offering a novel candidate therapeutic option to counteract skeletal and cardiac muscle injury.