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.

Epigenetic disorders: Lessons from the animals-animal models in chromatinopathies.

Chromatinopathies are defined as genetic disorders caused by mutations in genes coding for protein involved in the chromatin state balance. So far 82 human conditions have been described belonging to this group of congenital disorders, sharing some molecular features and clinical signs. For almost all of these conditions, no specific treatment is available. For better understanding the molecular cascade caused by chromatin imbalance and for envisaging possible therapeutic strategies it is fundamental to combine clinical and basic research studies. To this end, animal modelling systems represent an invaluable tool to study chromatinopathies. In this review, we focused on available data in the literature of animal models mimicking the human genetic conditions. Importantly, affected organs and abnormalities are shared in the different animal models and most of these abnormalities are reported as clinical manifestation, underlying the parallelism between clinics and translational research.

KMT2A: Umbrella Gene for Multiple Diseases.

KMT2A (Lysine methyltransferase 2A) is a member of the epigenetic machinery, encoding a lysine methyltransferase responsible for the transcriptional activation through lysine 4 of histone 3 (H3K4) methylation. KMT2A has a crucial role in gene expression, thus it is associated to pathological conditions when found mutated. KMT2A germinal mutations are associated to Wiedemann-Steiner syndrome and also in patients with initial clinical diagnosis of several other chromatinopathies (i.e., Coffin-Siris syndromes, Kabuki syndrome, Cornelia De Lange syndrome, Rubinstein-Taybi syndrome), sharing an overlapping phenotype. On the other hand, KMT2A somatic mutations have been reported in several tumors, mainly blood malignancies. Due to its evolutionary conservation, the role of KMT2A in embryonic development, hematopoiesis and neurodevelopment has been explored in different animal models, and in recent decades, epigenetic treatments for disorders linked to KMT2A dysfunction have been extensively investigated. To note, pharmaceutical compounds acting on tumors characterized by KMT2A mutations have been formulated, and even nutritional interventions for chromatinopathies have become the object of study due to the role of microbiota in epigenetic regulation.

Identical EP300 variant leading to Rubinstein-Taybi syndrome with different clinical and immunologic phenotype.

The Rubinstein-Taybi syndrome (RSTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, intellectual disability, growth deficiency, and recurrent infections. Mutations in the cyclic adenosine monophosphate response element-binding protein (CREB)-binding protein (CREBBP) or in the E1A-associated protein p300 (EP300) genes have been demonstrated in 55% (RSTS1) and up to 8% of the patients (RSTS2), respectively. Dysfunction of immune response has been reported in a subgroup of individuals with RSTS. Here we characterize two patients carrying the same EP300 variant and distinctive RSTS features (including congenital heart abnormalities, short stature, feeding problems, and gastroesophageal reflux). Whole exome sequencing did not support a dual molecular diagnosis hypothesis. Nonetheless, patients showed distinct clinical manifestations and immunological features. The most severe phenotype was associated with reduced T-cell production and diversity. This latter feature was confirmed in a control group of four RSTS patients.

Chromatin Imbalance as the Vertex Between Fetal Valproate Syndrome and Chromatinopathies.

Prenatal exposure to valproate (VPA), an antiepileptic drug, has been associated with fetal valproate spectrum disorders (FVSD), a clinical condition including congenital malformations, developmental delay, intellectual disability as well as autism spectrum disorder, together with a distinctive facial appearance. VPA is a known inhibitor of histone deacetylase which regulates the chromatin state. Interestingly, perturbations of this epigenetic balance are associated with chromatinopathies, a heterogeneous group of Mendelian disorders arising from mutations in components of the epigenetic machinery. Patients affected from these disorders display a plethora of clinical signs, mainly neurological deficits and intellectual disability, together with distinctive craniofacial dysmorphisms. Remarkably, critically examining the phenotype of FVSD and chromatinopathies, they shared several overlapping features that can be observed despite the different etiologies of these disorders, suggesting the possible existence of a common perturbed mechanism(s) during embryonic development.

Insights into the Role of the Microbiota and of Short-Chain Fatty Acids in Rubinstein-Taybi Syndrome.

The short-chain fatty acid butyrate, produced by the gut microbiota, acts as a potent histone deacetylase (HDAC) inhibitor. We assessed possible ameliorative effects of butyrate, relative to other HDAC inhibitors, in in vitro and in vivo models of Rubinstein-Taybi syndrome (RSTS), a severe neurodevelopmental disorder caused by variants in the genes encoding the histone acetyltransferases CBP and p300. In RSTS cell lines, butyrate led to the patient-specific rescue of acetylation defects at subtoxic concentrations. Remarkably, we observed that the commensal gut microbiota composition in a cohort of RSTS patients is significantly depleted in butyrate-producing bacteria compared to healthy siblings. We demonstrate that the effects of butyrate and the differences in microbiota composition are conserved in a Drosophila melanogaster mutant for CBP, enabling future dissection of the gut-host interactions in an in vivo RSTS model. This study sheds light on microbiota composition in a chromatinopathy, paving the way for novel therapeutic interventions.

Lithium as a possible therapeutic strategy for Cornelia de Lange syndrome.

Cornelia de Lange Syndrome (CdLS) is a rare developmental disorder affecting a multitude of organs including the central nervous system, inducing a variable neurodevelopmental delay. CdLS malformations derive from the deregulation of developmental pathways, inclusive of the canonical WNT pathway. We have evaluated MRI anomalies and behavioral and neurological clinical manifestations in CdLS patients. Importantly, we observed in our cohort a significant association between behavioral disturbance and structural abnormalities in brain structures of hindbrain embryonic origin. Considering the cumulative evidence on the cohesin-WNT-hindbrain shaping cascade, we have explored possible ameliorative effects of chemical activation of the canonical WNT pathway with lithium chloride in different models: (I) Drosophila melanogaster CdLS model showing a significant rescue of mushroom bodies morphology in the adult flies; (II) mouse neural stem cells restoring physiological levels in proliferation rate and differentiation capabilities toward the neuronal lineage; (III) lymphoblastoid cell lines from CdLS patients and healthy donors restoring cellular proliferation rate and inducing the expression of CyclinD1. This work supports a role for WNT-pathway regulation of CdLS brain and behavioral abnormalities and a consistent phenotype rescue by lithium in experimental models.

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.

Generation of the Becker muscular dystrophy patient derived induced pluripotent stem cell line carrying the DMD splicing mutation c.1705-8 T>C.

Becker Muscular dystrophy (BMD) is an X-linked syndrome characterized by progressive muscle weakness. BMD is generally less severe than Duchenne Muscular Dystrophy. BMD is caused by mutations in the dystrophin gene that normally give rise to the production of a truncated but partially functional dystrophin protein. We generated an induced pluripotent cell line from dermal fibroblasts of a BMD patient carrying a splice mutation in the dystrophin gene (c.1705-8 T>C). The iPSC cell-line displayed the characteristic pluripotent-like morphology, expressed pluripotency markers, differentiated into cells of the three germ layers and had a normal karyotype.

Customised next-generation sequencing multigene panel to screen a large cohort of individuals with chromatin-related disorder.

BACKGROUND: The regulation of the chromatin state by epigenetic mechanisms plays a central role in gene expression, cell function, and maintenance of cell identity. Hereditary disorders of chromatin regulation are a group of conditions caused by abnormalities of the various components of the epigenetic machinery, namely writers, erasers, readers, and chromatin remodelers. Although neurological dysfunction is almost ubiquitous in these disorders, the constellation of additional features characterizing many of these genes and the emerging clinical overlap among them indicate the existence of a community of syndromes. The introduction of high-throughput next generation sequencing (NGS) methods for testing multiple genes simultaneously is a logical step for the implementation of diagnostics of these disorders. METHODS: We screened a heterogeneous cohort of 263 index patients by an NGS-targeted panel, containing 68 genes associated with more than 40 OMIM entries affecting chromatin function. RESULTS: This strategy allowed us to identify clinically relevant variants in 87 patients (32%), including 30 for which an alternative clinical diagnosis was proposed after sequencing analysis and clinical re-evaluation. CONCLUSION: Our findings indicate that this approach is effective not only in disorders with locus heterogeneity, but also in order to anticipate unexpected misdiagnoses due to clinical overlap among cognate disorders. Finally, this work highlights the utility of a prompt diagnosis in such a clinically and genetically heterogeneous group of disorders that we propose to group under the umbrella term of chromatinopathies.

Chromatinopathies: A focus on Cornelia de Lange syndrome.

In recent years, many genes have been associated with chromatinopathies classified as "Cornelia de Lange Syndrome-like." It is known that the phenotype of these patients becomes less recognizable, overlapping to features characteristic of other syndromes caused by genetic variants affecting different regulators of chromatin structure and function. Therefore, Cornelia de Lange syndrome diagnosis might be arduous due to the seldom discordance between unexpected molecular diagnosis and clinical evaluation. Here, we review the molecular features of Cornelia de Lange syndrome, supporting the hypothesis that "CdLS-like syndromes" are part of a larger "rare disease family" sharing multiple clinical features and common disrupted molecular pathways.

Exploring by whole exome sequencing patients with initial diagnosis of Rubinstein-Taybi syndrome: the interconnections of epigenetic machinery disorders.

Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant neurodevelopmental disease affecting 1:125,000 newborns characterized by intellectual disability, growth retardation, facial dysmorphisms and skeletal abnormalities. RSTS is caused by mutations in genes encoding for writers of the epigenetic machinery: CREBBP (~ 60%) or its homologous EP300 (~ 10%). No causative mutation is identified in up to 30% of patients. We performed whole-exome sequencing (WES) on eight RSTS-like individuals who had normal high-resolution array CGH testing and were CREBBP- and EP300-mutation -negative, to identify the molecular cause. In four cases, we identified putatively causal variants in three genes (ASXL1, KMT2D and KMT2A) encoding members of the epigenetic machinery known to be associated with the Bohring-Opitz, Kabuki and Wiedemann-Steiner syndromes. Each variant is novel, de novo, fulfills the ACMG criteria and is predicted to result in loss-of-function leading to haploinsufficiency of the epi-gene. In two of the remaining cases, homozygous/compound heterozygous variants in XYLT2 and PLCB4 genes, respectively, associated with spondyloocular and auriculocondylar 2 syndromes and in the latter an additional candidate variant in XRN2, a gene yet unrelated to any disease, were detected, but their pathogenicity remains uncertain. These results underscore the broad clinical spectrum of Mendelian disorders of the epigenetic apparatus and the high rate of WES disclosure of the genetic basis in cases which may pose a challenge for phenotype encompassing distinct syndromes. The overlapping features of distinct intellectual disability syndromes reflect common pathogenic molecular mechanisms affecting the complex regulation of balance between open and closed chromatin.

Insights into Mutation Effect in Three Poikiloderma with Neutropenia Patients by Transcript Analysis and Disease Evolution of Reported Patients with the Same Pathogenic Variants.

PURPOSE: Poikiloderma with neutropenia (PN) is a genodermatosis currently described in 77 patients, all presenting with early-onset poikiloderma, neutropenia, and several additional signs. Biallelic loss-of-function mutations in USB1 gene are detected in all molecularly tested patients but genotype-phenotype correlation remains elusive. Cancer predisposition is recognized among PN features and pathogenic variants found in patients who developed early in life myelodysplasia (n = 12), acute myeloid leukemia (n = 2), and squamous cell carcinoma (n = 2) should be kept into account in management and follow-up of novel patients. This will hopefully allow achieving data clustered on specific mutations relevant to oncological surveillance of the carrier patients. METHODS: We describe the clinical features of three unreported PN patients and characterize their USB1 pathogenic variants by transcript analysis to get insights into the effect on the overall phenotype and disease evolution. RESULTS: A Turkish boy is homozygous for the c.531delA deletion, a recurrent mutation in Turkey; an adult Italian male is compound heterozygous for two nonsense mutations, c.243G>A and c.541C>T, while an Italian boy is homozygous for the splicing c.683_693+1del variant. The identified mutations have already been reported in PN patients who developed hematologic or skin cancer. Aberrant mRNAs of all four mutated alleles could be identified confirming that transcripts of USB1 main isoform either carrying stop codons or mis-spliced may at least partially escape nonsense-mediated decay. CONCLUSIONS: Our study addresses the need of gathering insights on genotype-phenotype correlations in newly described PN patients, by transcript analysis and information on disease evolution of reported patients with the same pathogenic variants.