KBG Syndrome in 16 Indian Individuals.

We aimed to describe the clinical and genetic characteristics of 16 individuals with KBG syndrome (KBGS) from 13 Indian families. We retrospectively analyzed the clinical details of individuals with KBGS harboring a likely pathogenic/pathogenic variant in ANKRD11. We also analyzed their facial gestalt using Face2Gene and recorded the top three differential disorders suggested by the application. The most frequent clinical features observed in our cohort were as follows: learning and intellectual disability-14/15 (93%), skeletal abnormalities-14/15 (93%), postnatal short stature-13/15 (87%), brachydactyly-11/15 (73%), and characteristic facial appearance-13/15 (87%). We identified 12 single nucleotide variants (SNVs), including six recurrent and six novel variants, and a copy number variant in the 16q24.3 region encompassing ANKRD11 gene. The novel variants were as follows: p.(Gln1236Ter), p.(Asp884ThrfsTer93), p.(Arg1466GlyfsTer87), p.(Tyr2056Ter), p.(Leu955TrpfsTer22), and p.(Lys766ArgfsTer10). The identified SNVs in ANKRD11 clustered around exon 9. We observed a high concordance of Face2Gene in predicting KBGS.

DNA methylation episignature testing improves molecular diagnosis of Mendelian chromatinopathies.

PURPOSE: Chromatinopathies include more than 50 disorders caused by disease-causing variants of various components of chromatin structure and function. Many of these disorders exhibit unique genome-wide DNA methylation profiles, known as episignatures. In this study, the methylation profile of a large cohort of individuals with chromatinopathies was analyzed for episignature detection. METHODS: DNA methylation data was generated on extracted blood samples from 129 affected individuals with the Illumina Infinium EPIC arrays and analyzed using an established bioinformatic pipeline. RESULTS: The DNA methylation profiles matched and confirmed the sequence findings in both the discovery and validation cohorts. Twenty-five affected individuals carrying a variant of uncertain significance, did not show a methylation profile matching any of the known episignatures. Three additional variant of uncertain significance cases with an identified KDM6A variant were re-classified as likely pathogenic (n = 2) or re-assigned as Wolf-Hirschhorn syndrome (n = 1). Thirty of the 33 Next Generation Sequencing negative cases did not match a defined episignature while three matched Kabuki syndrome, Rubinstein-Taybi syndrome and BAFopathy respectively. CONCLUSION: With the expanding clinical utility of the EpiSign assay, DNA methylation analysis should be considered part of the testing cascade for individuals presenting with clinical features of Mendelian chromatinopathy disorders.

ANKRD11 variants: KBG syndrome and beyond.

Mutations affecting the transcriptional regulator Ankyrin Repeat Domain 11 (ANKRD11) are mainly associated with the multisystem developmental disorder known as KBG syndrome, but have also been identified in individuals with Cornelia de Lange syndrome (CdLS) and other developmental disorders caused by variants affecting different chromatin regulators. The extensive functional overlap of these proteins results in shared phenotypical features, which complicate the assessment of the clinical diagnosis. Additionally, re-evaluation of individuals at a later age occasionally reveals that the initial phenotype has evolved toward clinical features more reminiscent of a developmental disorder different from the one that was initially diagnosed. For this reason, variants in ANKRD11 can be ascribed to a broader class of disorders that fall within the category of the so-called chromatinopathies. In this work, we report on the clinical characterization of 23 individuals with variants in ANKRD11. The subjects present primarily with developmental delay, intellectual disability and dysmorphic features, and all but two received an initial clinical diagnosis of either KBG syndrome or CdLS. The number and the severity of the clinical signs are overlapping but variable and result in a broad spectrum of phenotypes, which could be partially accounted for by the presence of additional molecular diagnoses and distinct pathogenic mechanisms.

A novel patient with White-Sutton syndrome refines the mutational and clinical repertoire of the POGZ-related phenotype and suggests further observations.

A rare developmental delay (DD)/intellectual disability (ID) syndrome with craniofacial dysmorphisms and autistic features, termed White-Sutton syndrome (WHSUS, MIM#614787), has been recently described, identifying truncating mutations in the chromatin regulator POGZ (KIAA0461, MIM#614787). We describe a further WHSUS patient harboring a novel nonsense de novo POGZ variant, which afflicts a protein domain with transposase activity less frequently impacted by mutational events (DDE domain). This patient displays additional physical and behavioral features, these latter mimicking Smith-Magenis syndrome (SMS, MIM#182290). Considering sleep-wake cycle anomalies and abnormal behavior manifested by this boy, we reinforced the clinical resemblance between WHSUS and SMS, being both chromatinopathies. In addition, using the DeepGestalt technology, we identified a different facial overlap between WHSUS patients with mutations in the DDE domain (Group 1) and individuals harboring variants in other protein domains/regions (Group 2). This report further delineates the clinical and molecular repertoire of the POGZ-related phenotype, adding a novel patient with uncommon clinical and behavioral features and provides the first computer-aided facial study of WHSUS patients.

Intrauterine growth in chromatinopathies: A long road for better understanding and for improving clinical management.

BACKGROUND: Chromatinopathies are a heterogeneous group of genetic disorders caused by pathogenic variants in genes coding for chromatin state balance proteins. Remarkably, many of these syndromes present unbalanced postnatal growth, both under- and over-, although little has been described in the literature. Fetal growth measurements are common practice in pregnancy management and values within normal ranges indicate proper intrauterine growth progression; on the contrary, abnormalities in intrauterine fetal growth open the discussion of possible pathogenesis affecting growth even in the postnatal period. METHODS: Among the numerous chromatinopathies, we have selected six of the most documented in the literature offering evidence about two fetal overgrowth (Sotos and Weaver syndrome) and four fetal undergrowth syndromes (Bohring Opitz, Cornelia de Lange, Floating-Harbor, and Meier Gorlin syndrome), describing their molecular characteristics, maternal biochemical results and early pregnancy findings, prenatal ultrasound findings, and postnatal characteristics. RESULTS/CONCLUSION: To date, the scarce data in the literature on prenatal findings are few and inconclusive, even though these parameters may contribute to a more rapid and accurate diagnosis, calling for a better and more detailed description of pregnancy findings.

Epigenetic regulation of autophagy-related genes: Implications for neurodevelopmental disorders.

Macroautophagy/autophagy is an evolutionarily highly conserved catabolic process that is important for the clearance of cytosolic contents to maintain cellular homeostasis and survival. Recent findings point toward a critical role for autophagy in brain function, not only by preserving neuronal health, but especially by controlling different aspects of neuronal development and functioning. In line with this, mutations in autophagy-related genes are linked to various key characteristics and symptoms of neurodevelopmental disorders (NDDs), including autism, micro-/macrocephaly, and epilepsy. However, the group of NDDs caused by mutations in autophagy-related genes is relatively small. A significant proportion of NDDs are associated with mutations in genes encoding epigenetic regulatory proteins that modulate gene expression, so-called chromatinopathies. Intriguingly, several of the NDD-linked chromatinopathy genes have been shown to regulate autophagy-related genes, albeit in non-neuronal contexts. From these studies it becomes evident that tight transcriptional regulation of autophagy-related genes is crucial to control autophagic activity. This opens the exciting possibility that aberrant autophagic regulation might underly nervous system impairments in NDDs with disturbed epigenetic regulation. We here summarize NDD-related chromatinopathy genes that are known to regulate transcriptional regulation of autophagy-related genes. Thereby, we want to highlight autophagy as a candidate key hub mechanism in NDD-related chromatinopathies.Abbreviations: ADNP: activity dependent neuroprotector homeobox; ASD: autism spectrum disorder; ATG: AutTophaGy related; CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; EHMT: euchromatic histone lysine methyltransferase; EP300: E1A binding protein p300; EZH2: enhancer of zeste 2 polycomb repressive complex 2 subunit; H3K4me3: histone 3 lysine 4 trimethylation; H3K9me1/2/3: histone 3 lysine 9 mono-, di-, or trimethylation; H3K27me2/3: histone 3 lysine 27 di-, or trimethylation; hiPSCs: human induced pluripotent stem cells; HSP: hereditary spastic paraplegia; ID: intellectual disability; KANSL1: KAT8 regulatory NSL complex subunit 1; KAT8: lysine acetyltransferase 8; KDM1A/LSD1: lysine demethylase 1A; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; NDD: neurodevelopmental disorder; PHF8: PHD finger protein 8; PHF8-XLID: PHF8-X linked intellectual disability syndrome; PTM: post-translational modification; SESN2: sestrin 2; YY1: YY1 transcription factor; YY1AP1: YY1 associated protein 1.

The overlapping of phenotypes in Wiedemann-Steiner, Kleefstra and Coffin-Siris syndromes: a study of eleven patients.

BACKGROUND: Some chromatinopathies may present with common clinical findings (intellectual disability, brain and limb malformation, facial dysmorphism). Furthermore, one of their cardinal shared features is growth dysregulation.We aimed to assess and deepen this resemblance in three specific conditions, namely Wiedemann-Steiner (WDSTS), Kleefstra (KLEFS1) and Coffin-Siris syndrome (CSS1), with a particular focus on possible metabolic roots. METHODS: Eleven patients were enrolled, three with WDSTS, five with KLEFS1 and three with CSS1, referring to Fondazione IRCCS Ca' Granda Ospedale Maggiore, Milan, Italy. We performed both a physical examination with detailed anthropometric measurements and an evaluation of the patients' REE (rest energy expenditure) by indirect calorimetry, comparing the results with age- and sex-matched healthy controls. RESULTS: We observed new clinical features and overlap between these conditions suggesting that different disturbances of epigenetic machinery genes can converge on a common effect, leading to overlapping clinical phenotypes.The REE was not distinguishable between the three conditions and healthy controls. CONCLUSIONS: Epigenetic machinery plays an essential role both in growth regulation and in neurodevelopment; we recommend evaluating skeletal [craniovertebral junction abnormalities (CVJ) polydactyly], otolaryngological [obstructive sleep apnea syndrome (OSAs), recurrent otitis media], dental [tooth agenesis, talon cusps], and central nervous system (CNS) [olfactory bulbs and cerebellum anomalies] features. These features could be included in monitoring guidelines. Further studies are needed to deepen the knowledge about energy metabolism.

KAT6A mutations in Arboleda-Tham syndrome drive epigenetic regulation of posterior HOXC cluster.

Arboleda-Tham Syndrome (ARTHS) is a rare genetic disorder caused by heterozygous, de novo truncating mutations in Lysine(K) acetyltransferase 6A (KAT6A). ARTHS is clinically heterogeneous and characterized by several common features including intellectual disability, developmental and speech delay, hypotonia and affects multiple organ systems. KAT6A is highly expressed in early development and plays a key role in cell-type specific differentiation. KAT6A is the enzymatic core of a histone-acetylation protein complex, however the direct histone targets and gene regulatory effects remain unknown. In this study, we use ARTHS patient (n=8) and control (n=14) dermal fibroblasts and perform comprehensive profiling of the epigenome and transcriptome caused by KAT6A mutations. We identified differential chromatin accessibility within the promoter or gene body of 23%(14/60) of genes that were differentially expressed between ARTHS and controls. Within fibroblasts, we show a distinct set of genes from the posterior HOXC gene cluster (HOXC10, HOXC11, HOXC-AS3, HOXC-AS2, HOTAIR) that are overexpressed in ARTHS and are transcription factors critical for early development body segment patterning. The genomic loci harboring HOXC genes are epigenetically regulated with increased chromatin accessibility, high levels of H3K23ac, and increased gene-body DNA methylation compared to controls, all of which are consistent with transcriptomic overexpression. Finally, we used unbiased proteomic mass spectrometry and identified two new histone post-translational modifications (PTMs) that are disrupted in ARTHS: H2A and H3K56 acetylation. Our multi-omics assays have identified novel histone and gene regulatory roles of KAT6A in a large group of ARTHS patients harboring diverse pathogenic mutations. This work provides insight into the role of KAT6A on the epigenomic regulation in somatic cell types.

Success and Pitfalls of Genetic Testing in Undiagnosed Diseases: Whole Exome Sequencing and Beyond.

Novel approaches to uncover the molecular etiology of neurodevelopmental disorders (NDD) are highly needed. Even using a powerful tool such as whole exome sequencing (WES), the diagnostic process may still prove long and arduous due to the high clinical and genetic heterogeneity of these conditions. The main strategies to improve the diagnostic rate are based on family segregation, re-evaluation of the clinical features by reverse-phenotyping, re-analysis of unsolved NGS-based cases and epigenetic functional studies. In this article, we described three selected cases from a cohort of patients with NDD in which trio WES was applied, in order to underline the typical challenges encountered during the diagnostic process: (1) an ultra-rare condition caused by a missense variant in MEIS2, identified through the updated Solve-RD re-analysis; (2) a patient with Noonan-like features in which the NGS analysis revealed a novel variant in NIPBL causing Cornelia de Lange syndrome; and (3) a case with de novo variants in genes involved in the chromatin-remodeling complex, for which the study of the epigenetic signature excluded a pathogenic role. In this perspective, we aimed to (i) provide an example of the relevance of the genetic re-analysis of all unsolved cases through network projects on rare diseases; (ii) point out the role and the uncertainties of the reverse phenotyping in the interpretation of the genetic results; and (iii) describe the use of methylation signatures in neurodevelopmental syndromes for the validation of the variants of uncertain significance.

Chromatin dynamics in human brain development and disease.

Chromatin-related genes are frequently mutated in neurodevelopmental disorders; yet, the mechanisms by which these perturbations disrupt brain assembly and function are not understood. Here, we describe how recent advances in transcriptional and chromatin profiling in combination with cellular models are beginning to inform our understanding of neurodevelopment and chromatinopathies.

Chromatin Structure and Dynamics: Focus on Neuronal Differentiation and Pathological Implication.

Chromatin structure is an essential regulator of gene expression. Its state of compaction contributes to the regulation of genetic programs, in particular during differentiation. Epigenetic processes, which include post-translational modifications of histones, DNA methylation and implication of non-coding RNA, are powerful regulators of gene expression. Neurogenesis and neuronal differentiation are spatio-temporally regulated events that allow the formation of the central nervous system components. Here, we review the chromatin structure and post-translational histone modifications associated with neuronal differentiation. Studying the impact of histone modifications on neuronal differentiation improves our understanding of the pathophysiological mechanisms of chromatinopathies and opens up new therapeutic avenues. In addition, we will discuss techniques for the analysis of histone modifications on a genome-wide scale and the pathologies associated with the dysregulation of the epigenetic machinery.

Following Excitation/Inhibition Ratio Homeostasis from Synapse to EEG in Monogenetic Neurodevelopmental Disorders.

Pharmacological options for neurodevelopmental disorders are limited to symptom suppressing agents that do not target underlying pathophysiological mechanisms. Studies on specific genetic disorders causing neurodevelopmental disorders have elucidated pathophysiological mechanisms to develop more rational treatments. Here, we present our concerted multi-level strategy 'BRAINMODEL', focusing on excitation/inhibition ratio homeostasis across different levels of neuroscientific interrogation. The aim is to develop personalized treatment strategies by linking iPSC-based models and novel EEG measurements to patient report outcome measures in individual patients. We focus our strategy on chromatin- and SNAREopathies as examples of severe genetic neurodevelopmental disorders with an unmet need for rational interventions.

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.

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.

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.

Cornelia de Lange Syndrome as Paradigm of Chromatinopathies.

Chromatinopathies can be defined as a class of neurodevelopmental disorders caused by mutations affecting proteins responsible for chromatin remodeling and transcriptional regulation. The resulting dysregulation of gene expression favors the onset of a series of clinical features such as developmental delay, intellectual disability, facial dysmorphism, and behavioral disturbances. Cornelia de Lange syndrome (CdLS) is a prime example of a chromatinopathy. It is caused by mutations affecting subunits or regulators of the cohesin complex, a multisubunit protein complex involved in various molecular mechanisms such as sister chromatid cohesion, transcriptional regulation and formation of topologically associated domains. However, disease-causing variants in non-cohesin genes with overlapping functions have also been described in association with CdLS. Notably, the majority of these genes had been previously found responsible for distinct neurodevelopmental disorders that also fall within the category of chromatinopathies and are frequently considered as differential diagnosis for CdLS. In this review, we provide a systematic overview of the current literature to summarize all mutations in non-cohesin genes identified in association with CdLS phenotypes and discuss about the interconnection of proteins belonging to the chromatinopathies network.

Rubinstein-Taybi Syndrome: A Model of Epigenetic Disorder.

The Rubinstein-Taybi syndrome (RSTS) is a rare congenital developmental disorder characterized by a typical facial dysmorphism, distal limb abnormalities, intellectual disability, and many additional phenotypical features. It occurs at between 1/100,000 and 1/125,000 births. Two genes are currently known to cause RSTS, CREBBP and EP300, mutated in around 55% and 8% of clinically diagnosed cases, respectively. To date, 500 pathogenic variants have been reported for the CREBBP gene and 118 for EP300. These two genes encode paralogs acting as lysine acetyltransferase involved in transcriptional regulation and chromatin remodeling with a key role in neuronal plasticity and cognition. Because of the clinical heterogeneity of this syndrome ranging from the typical clinical diagnosis to features overlapping with other Mendelian disorders of the epigenetic machinery, phenotype/genotype correlations remain difficult to establish. In this context, the deciphering of the patho-physiological process underlying these diseases and the definition of a specific episignature will likely improve the diagnostic efficiency but also open novel therapeutic perspectives. This review summarizes the current clinical and molecular knowledge and highlights the epigenetic regulation of RSTS as a model of chromatinopathy.

Analysis of a Set of KDM5C Regulatory Genes Mutated in Neurodevelopmental Disorders Identifies Temporal Coexpression Brain Signatures.

Dysregulation of transcriptional pathways is observed in multiple forms of neurodevelopmental disorders (NDDs), such as intellectual disability (ID), epilepsy and autism spectrum disorder (ASD). We previously demonstrated that the NDD genes encoding lysine-specific demethylase 5C (KDM5C) and its transcriptional regulators Aristaless related-homeobox (ARX), PHD Finger Protein 8 (PHF8) and Zinc Finger Protein 711 (ZNF711) are functionally connected. Here, we show their relation to each other with respect to the expression levels in human and mouse datasets and in vivo mouse analysis indicating that the coexpression of these syntenic X-chromosomal genes is temporally regulated in brain areas and cellular sub-types. In co-immunoprecipitation assays, we found that the homeotic transcription factor ARX interacts with the histone demethylase PHF8, indicating that this transcriptional axis is highly intersected. Furthermore, the functional impact of pathogenic mutations of ARX, KDM5C, PHF8 and ZNF711 was tested in lymphoblastoid cell lines (LCLs) derived from children with varying levels of syndromic ID establishing the direct correlation between defects in the KDM5C-H3K4me3 pathway and ID severity. These findings reveal novel insights into epigenetic processes underpinning NDD pathogenesis and provide new avenues for assessing developmental timing and critical windows for potential treatments.

Syndromic Craniosynostosis Can Define New Candidate Genes for Suture Development or Result from the Non-specifc Effects of Pleiotropic Genes: Rasopathies and Chromatinopathies as Examples.

Craniosynostosis is a heterogeneous condition caused by the premature fusion of cranial sutures, occurring mostly as an isolated anomaly. Pathogenesis of non-syndromic forms of craniosynostosis is largely unknown. In about 15-30% of cases craniosynostosis occurs in association with other physical anomalies and it is referred to as syndromic craniosynostosis. Syndromic forms of craniosynostosis arise from mutations in genes belonging to the Fibroblast Growth Factor Receptor (FGFR) family and the interconnected molecular pathways in most cases. However it can occur in association with other gene variants and with a variety of chromosome abnormalities as well, usually in association with intellectual disability (ID) and additional physical anomalies. Evaluating the molecular properties of the genes undergoing intragenic mutations or copy number variations (CNVs) along with prevalence of craniosynostosis in different conditions and animal models if available, we made an attempt to define two distinct groups of unusual syndromic craniosynostosis, which can reflect direct effects of emerging new candidate genes with roles in suture homeostasis or a non-specific phenotypic manifestation of pleiotropic genes, respectively. RASopathies and 9p23p22.3 deletions are reviewed as examples of conditions in the first group. In particular, we found that craniosynostosis is a relatively common component manifestation of cardio-facio-cutaneous (CFC) syndrome. Chromatinopathies and neurocristopathies are presented as examples of conditions in the second group. We observed that craniosynostosis is uncommon on average in these conditions. It was randomly associated with Kabuki, Koolen-de Vries/KANSL1 haploinsufficiency and Mowat-Wilson syndromes and in KAT6B-related disorders. As an exception, trigonocephaly in Bohring-Opitz syndrome reflects specific molecular properties of the chromatin modifier ASXL1 gene. Surveillance for craniosynostosis in syndromic forms of intellectual disability, as well as ascertainment of genomic CNVs by array-CGH in apparently non-syndromic craniosynostosis is recommended, to allow for improvement of both the clinical outcome of patients and the accurate individual diagnosis.