Missense variants causing Wiedemann-Steiner syndrome preferentially occur in the KMT2A-CXXC domain and are accurately classified using AlphaFold2

Funding Information: This work was supported by a grant from the Wiedemann-Steiner Foundation to HTB (salary coverage of TR). HTB is also supported by the Louma G. Foundation, the Icelandic Research Fund (#217988, #195835, #206806) and the Icelandic Technology Development Fund (#2010588). Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (#R01GM121459 to LB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Publisher Copyright: © 2022 Reynisdottir et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Wiedemann-Steiner syndrome (WDSTS) is a neurodevelopmental disorder caused by de novo variants in KMT2A, which encodes a multi-domain histone methyltransferase. To gain insight into the currently unknown pathogenesis of WDSTS, we examined the spatial distribution of likely WDSTS-causing variants across the 15 different domains of KMT2A. Compared to variants in healthy controls, WDSTS variants exhibit a 61.9-fold overrepresentation within the CXXC domain–which mediates binding to unmethylated CpGs–suggesting a major role for this domain in mediating the phenotype. In contrast, we find no significant overrepresentation within the catalytic SET domain. Corroborating these results, we find that hippocampal neurons from Kmt2a-deficient mice demonstrate disrupted histone methylation (H3K4me1 and H3K4me3) preferentially at CpG-rich regions, but this has no systematic impact on gene expression. Motivated by these results, we combine accurate prediction of the CXXC domain structure by AlphaFold2 with prior biological knowledge to develop a classification scheme for missense variants in the CXXC domain. Our classifier achieved 92.6% positive and 92.9% negative predictive value on a hold-out test set. This classification performance enabled us to subsequently perform an in silico saturation mutagenesis and classify a total of 445 variants according to their functional effects. Our results yield a novel insight into the mechanistic basis of WDSTS and provide an example of how AlphaFold2 can contribute to the in silico characterization of variant effects with very high accuracy, suggesting a paradigm potentially applicable to many other Mendelian disorders.Peer reviewe

Leveraging large-scale datasets to understand the interaction between the genome and the epigenome

Epigenetics is typically described as a layer of molecular information above and beyond the DNA sequence. While this conceptualization is certainly accurate to some extent, there is also a tight connection between the genome and the epigenome, as the basic components of the epigenetic machinery (EM) are DNA-encoded. This thesis focuses on four such genetic components: genes encoding for the proteins of the histone machinery, genes encoding for the proteins of the DNA methylation machinery, genes encoding for chromatin remodelers, and CpG dinucleotides. We first perform a systematic analysis of all human EM genes, and characterize them with respect to their tolerance to variation, both at the whole-gene level, and the local, protein domain level. We then discover a systems-level property (co-expression), that is specifically exhibited by a large subset of variation-intolerant EM genes, and may be particularly relevant to their involvement in neurodevelopment. Finally, we shift our focus on the CpG dinucleotides. We show that a high promoter CpG density is not merely a generic feature of human promoters, but is preferentially encountered at the promoters of the most loss-of-function intolerant genes. This coupling calls into question the prevailing view that CpG islands are not subject to selection. It also has practical utility, as it allows us to train a simple and easily interpretable predictive model of loss-of-function intolerance that outperforms existing predictors and classifies 1,760 genes - which are currently unascertained - as highly loss-of-function-intolerant or not. Together, the results presented in this thesis provide new insights into the interaction between the genome and the epigenome

Leveraging large-scale datasets to understand the interaction between the genome and the epigenome

Epigenetics is typically described as a layer of molecular information above and beyond the DNA sequence. While this conceptualization is certainly accurate to some extent, there is also a tight connection between the genome and the epigenome, as the basic components of the epigenetic machinery (EM) are DNA-encoded. This thesis focuses on four such genetic components: genes encoding for the proteins of the histone machinery, genes encoding for the proteins of the DNA methylation machinery, genes encoding for chromatin remodelers, and CpG dinucleotides. We first perform a systematic analysis of all human EM genes, and characterize them with respect to their tolerance to variation, both at the whole-gene level, and the local, protein domain level. We then discover a systems-level property (co-expression), that is specifically exhibited by a large subset of variation-intolerant EM genes, and may be particularly relevant to their involvement in neurodevelopment. Finally, we shift our focus on the CpG dinucleotides. We show that a high promoter CpG density is not merely a generic feature of human promoters, but is preferentially encountered at the promoters of the most loss-of-function intolerant genes. This coupling calls into question the prevailing view that CpG islands are not subject to selection. It also has practical utility, as it allows us to train a simple and easily interpretable predictive model of loss-of-function intolerance that outperforms existing predictors and classifies 1,760 genes - which are currently unascertained - as highly loss-of-function-intolerant or not. Together, the results presented in this thesis provide new insights into the interaction between the genome and the epigenome

Promoter CpG Density Predicts Downstream Gene Loss-of-Function Intolerance.

To access publisher's full text version of this article click on the hyperlink belowThe aggregation and joint analysis of large numbers of exome sequences has recently made it possible to derive estimates of intolerance to loss-of-function (LoF) variation for human genes. Here, we demonstrate strong and widespread coupling between genic LoF intolerance and promoter CpG density across the human genome. Genes downstream of the most CpG-rich promoters (top 10% CpG density) have a 67.2% probability of being highly LoF intolerant, using the LOEUF metric from gnomAD. This is in contrast to 7.4% of genes downstream of the most CpG-poor (bottom 10% CpG density) promoters. Combining promoter CpG density with exonic and promoter conservation explains 33.4% of the variation in LOEUF, and the contribution of CpG density exceeds the individual contributions of exonic and promoter conservation. We leverage this to train a simple and easily interpretable predictive model that outperforms other existing predictors and allows us to classify 1,760 genes-which are currently unascertained in gnomAD-as highly LoF intolerant or not. These predictions have the potential to aid in the interpretation of novel variants in the clinical setting. Moreover, our results reveal that high CpG density is not merely a generic feature of human promoters but is preferentially encountered at the promoters of the most selectively constrained genes, calling into question the prevailing view that CpG islands are not subject to selection. Keywords: CpG density; CpG islands; GC content; dosage sensitivity; epigenetics; gnomAD; haploinsufficiency; loss-of-function; promoters; selection.United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS) Maryland Genetics, Epidemiology, and Medicine (MD-GEM) training program - Burroughs Wellcome Fund Louma G. Foundatio

Coexpression patterns define epigenetic regulators associated with neurological dysfunction.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadCoding variants in epigenetic regulators are emerging as causes of neurological dysfunction and cancer. However, a comprehensive effort to identify disease candidates within the human epigenetic machinery (EM) has not been performed; it is unclear whether features exist that distinguish between variation-intolerant and variation-tolerant EM genes, and between EM genes associated with neurological dysfunction versus cancer. Here, we rigorously define 295 genes with a direct role in epigenetic regulation (writers, erasers, remodelers, readers). Systematic exploration of these genes reveals that although individual enzymatic functions are always mutually exclusive, readers often also exhibit enzymatic activity (dual-function EM genes). We find that the majority of EM genes are very intolerant to loss-of-function variation, even when compared to the dosage sensitive transcription factors, and we identify 102 novel EM disease candidates. We show that this variation intolerance is driven by the protein domains encoding the epigenetic function, suggesting that disease is caused by a perturbed chromatin state. We then describe a large subset of EM genes that are coexpressed within multiple tissues. This subset is almost exclusively populated by extremely variation-intolerant genes and shows enrichment for dual-function EM genes. It is also highly enriched for genes associated with neurological dysfunction, even when accounting for dosage sensitivity, but not for cancer-associated EM genes. Finally, we show that regulatory regions near epigenetic regulators are genetically important for common neurological traits. These findings prioritize novel disease candidate EM genes and suggest that this coexpression plays a functional role in normal neurological homeostasis.National Institute of General Medical Sciences of the National Institutes of Health Maryland Genetics, Epidemiology and Medicine (MD-GEM) training program - Burroughs-Wellcome Fund Johns Hopkins University Louma G. Foundation National Institutes of Health awards from the National Human Genome Research Institute National Institute of General Medical Science

Leveraging the Mendelian disorders of the epigenetic machinery to systematically map functional epigenetic variation.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadAlthough each Mendelian Disorder of the Epigenetic Machinery (MDEM) has a different causative gene, there are shared disease manifestations. We hypothesize that this phenotypic convergence is a consequence of shared epigenetic alterations. To identify such shared alterations, we interrogate chromatin (ATAC-seq) and expression (RNA-seq) states in B cells from three MDEM mouse models (Kabuki [KS] type 1 and 2 and Rubinstein-Taybi type 1 [RT1] syndromes). We develop a new approach for the overlap analysis and find extensive overlap primarily localized in gene promoters. We show that disruption of chromatin accessibility at promoters often disrupts downstream gene expression, and identify 587 loci and 264 genes with shared disruption across all three MDEMs. Subtle expression alterations of multiple, IgA-relevant genes, collectively contribute to IgA deficiency in KS1 and RT1, but not in KS2. We propose that the joint study of MDEMs offers a principled approach for systematically mapping functional epigenetic variation in mammals. Keywords: IgA deficiency; Mendelian; chromatin; computational biology; computational methods; epigenetics; genetics; genomics; histone machinery; mouse; systems biology.Louma G Foundation Icelandic Research Fund Icelandic Technology Development Fund Johns Hopkins University Maryland Genetics, Epidemiology and Medicine (MD-GEM) training program - Burroughs-Wellcome Fund United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS

Precocious chondrocyte differentiation disrupts skeletal growth in Kabuki syndrome mice.

To access publisher's full text version of this article click on the hyperlink belowKabuki syndrome 1 (KS1) is a Mendelian disorder of the epigenetic machinery caused by mutations in the gene encoding KMT2D, which methylates lysine 4 on histone H3 (H3K4). KS1 is characterized by intellectual disability, postnatal growth retardation, and distinct craniofacial dysmorphisms. A mouse model (Kmt2d+/βGeo) exhibits features of the human disorder and has provided insight into other phenotypes; however, the mechanistic basis of skeletal abnormalities and growth retardation remains elusive. Using high-resolution micro-CT, we show that Kmt2d+/βGeo mice have shortened long bones and ventral bowing of skulls. In vivo expansion of growth plates within skulls and long bones suggests disrupted endochondral ossification as a common disease mechanism. Stable chondrocyte cell lines harboring inactivating mutations in Kmt2d exhibit precocious differentiation, further supporting this mechanism. A known inducer of chondrogenesis, SOX9, and its targets show markedly increased expression in Kmt2d-/- chondrocytes. By transcriptome profiling, we identify Shox2 as a putative KMT2D target. We propose that decreased KMT2D-mediated H3K4me3 at Shox2 releases Sox9 inhibition and thereby leads to enhanced chondrogenesis, providing a potentially novel and plausible explanation for precocious chondrocyte differentiation. Our findings provide insight into the pathogenesis of growth retardation in KS1 and suggest therapeutic approaches for this and related disorders.Wellcome Trust Baltimore Center for Musculoskeletal Science 2015 Pilot and Feasibility Award William and Ella Owens Medical Research Foundation Johns Hopkins School of Medicine Clinician Scientist Award Hartwell Foundation Individual Biomedical Research Award United States Department of Health & Human Services National Institutes of Health (NIH) - USA Louma G. Foundatio

Precocious neuronal differentiation and disrupted oxygen responses in Kabuki syndrome.

To access publisher's full text version of this article click on the hyperlink belowChromatin modifiers act to coordinate gene expression changes critical to neuronal differentiation from neural stem/progenitor cells (NSPCs). Lysine-specific methyltransferase 2D (KMT2D) encodes a histone methyltransferase that promotes transcriptional activation and is frequently mutated in cancers and in the majority (>70%) of patients diagnosed with the congenital, multisystem intellectual disability disorder Kabuki syndrome 1 (KS1). Critical roles for KMT2D are established in various non-neural tissues, but the effects of KMT2D loss in brain cell development have not been described. We conducted parallel studies of proliferation, differentiation, transcription, and chromatin profiling in KMT2D-deficient human and mouse models to define KMT2D-regulated functions in neurodevelopmental contexts, including adult-born hippocampal NSPCs in vivo and in vitro. We report cell-autonomous defects in proliferation, cell cycle, and survival, accompanied by early NSPC maturation in several KMT2D-deficient model systems. Transcriptional suppression in KMT2D-deficient cells indicated strong perturbation of hypoxia-responsive metabolism pathways. Functional experiments confirmed abnormalities of cellular hypoxia responses in KMT2D-deficient neural cells and accelerated NSPC maturation in vivo. Together, our findings support a model in which loss of KMT2D function suppresses expression of oxygen-responsive gene programs important to neural progenitor maintenance, resulting in precocious neuronal differentiation in a mouse model of KS1.United States Department of Health & Human Services National Institutes of Health (NIH) - USA Icelandic Research Fund Louma G. Foundation United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD

Missense variants in the chromatin remodeler CHD1 are associated with neurodevelopmental disability

BackgroundThe list of Mendelian disorders of the epigenetic machinery has expanded rapidly during the last 5 years. A few missense variants in the chromatin remodeler CHD1 have been found in several large-scale sequencing efforts focused on uncovering the genetic aetiology of autism.ObjectivesTo explore whether variants in CHD1 are associated with a human phenotype.MethodsWe used GeneMatcher to identify other physicians caring for patients with variants in CHD1. We also explored the epigenetic consequences of one of these variants in cultured fibroblasts.ResultsHere we describe six CHD1 heterozygous missense variants in a cohort of patients with autism, speech apraxia, developmental delay and facial dysmorphic features. Importantly, three of these variants occurred de novo. We also report on a subject with a de novo deletion covering a large fraction of the CHD1 gene without any obvious neurological phenotype. Finally, we demonstrate increased levels of the closed chromatin modification H3K27me3 in fibroblasts from a subject carrying a de novo variant in CHD1.ConclusionsOur results suggest that variants in CHD1 can lead to diverse phenotypic outcomes; however, the neurodevelopmental phenotype appears to be limited to patients with missense variants, which is compatible with a dominant negative mechanism of disease