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Growth deficiency is a characteristic feature of both Kabuki syndrome 1 (KS1) and Kabuki syndrome 2 (KS2), Mendelian disorders of the epigenetic machinery with similar phenotypes but distinct genetic etiologies. We previously described skeletal growth deficiency in a mouse model of KS1 and further established that a Kmt2d-/- chondrocyte model of KS1 exhibits precocious differentiation. Here we characterized growth deficiency in a mouse model of KS2, Kdm6atm1d/+. We show that Kdm6atm1d/+ mice have decreased femur and tibia length compared to controls and exhibit abnormalities in cortical and trabecular bone structure. Kdm6atm1d/+ growth plates are also shorter, due to decreases in hypertrophic chondrocyte size and hypertrophic zone height. Given these disturbances in the growth plate, we generated Kdm6a-/- chondrogenic cell lines. Similar to our prior in vitro model of KS1, we found that Kdm6a-/- cells undergo premature, enhanced differentiation towards chondrocytes compared to Kdm6a+/+ controls. RNA-seq showed that Kdm6a-/- cells have a distinct transcriptomic profile that indicates dysregulation of cartilage development. Finally, we performed RNA-seq simultaneously on Kmt2d-/-, Kdm6a-/-, and control lines at Days 7 and 14 of differentiation. This revealed surprising resemblance in gene expression between Kmt2d-/- and Kdm6a-/- at both time points and indicates that the similarity in phenotype between KS1 and KS2 also exists at the transcriptional level., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests:HTB is a consultant for Mahzi therapeutics. The authors declare no other conflicts of interest relevant to this manuscript., (Copyright: © 2024 Gao 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.)

The multidisciplinary Epigenetics and Chromatin Clinic at Johns Hopkins provides comprehensive medical care for individuals with rare disorders that involve disrupted epigenetics. Initially centered on classical imprinting disorders, the focus shifted to the rapidly emerging group of genetic disorders resulting from pathogenic germline variants in epigenetic machinery genes. These are collectively called the Mendelian disorders of the epigenetic machinery (MDEMs), or more broadly, Chromatinopathies. In five years, 741 clinic visits have been completed for 432 individual patients, with 153 having confirmed epigenetic diagnoses. Of these, 115 individuals have one of 26 MDEMs with every single one exhibiting global developmental delay and/or intellectual disability. This supports prior observations that intellectual disability is the most common phenotypic feature of MDEMs. Additional common phenotypes in our clinic include growth abnormalities and neurodevelopmental issues, particularly hypotonia, attention-deficit/hyperactivity disorder (ADHD), and anxiety, with seizures and autism being less common. Overall, our patient population is representative of the broader group of MDEMs and includes mostly autosomal dominant disorders impacting writers more so than erasers, readers, and remodelers of chromatin marks. There is an increased representation of dual function components with a reader and an enzymatic domain. As expected, diagnoses were made mostly by sequencing but were aided in some cases by DNA methylation profiling. Our clinic has helped to facilitate the discovery of two new disorders, and our providers are actively developing and implementing novel therapeutic strategies for MDEMs. These data and our high follow-up rate of over 60% suggest that we are achieving our mission to diagnose, learn from, and provide optimal care for our patients with disrupted epigenetics., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Wiedemann-Steiner syndrome (WSS) is a neurogenetic disorder caused by heterozygous variants in KMT2A. Recent investigations suggest increased anxiety and behavior regulation challenges among those with WSS although the neurobehavioral phenotype remains largely unknown. This study aims to examine the pattern of and associations between executive functioning (EF) and behavior functioning among those with WSS., Method: This study involved utilizing caregiver-report inventories (Behavior Rating Inventory of Executive Function 2nd Edition, BRIEF-2; Adaptive Behavior Assessment 3rd Edition, ABAS-3; Strengths and Difficulties Questionnaire, SDQ) to assess day-to-day behavior functioning among those with WSS (N = 24; mean age = 10.68 years, SD = 3.19). Frequency of clinical elevations in daily difficulties in EF, adaptive behaviors, and behavior regulation were reported. Correlations and hierarchical linear regressions were used to determine the relationships between EF with behavior and adaptive functioning., Results: Out of our sample, 63% met clinical levels of executive functioning difficulties on the BRIEF-2, and 75% with Hyperactivity and 54% with Emotional Problems on the SDQ. In addition, 33% were rated >2 SD below the normative mean in overall adaptive functioning on the ABAS-3. Elevated ratings in BRIEF-2 Shift, reflective of challenges with mental flexibility, predicted more Emotional Problems and accounted for 33.5% of its variance. More difficulties in Emotional Control were related to greater adaptive deficits, accounting for 33.3% of its variance., Conclusions: Those with WSS are at risk for EF deficits, hyperactivity, and emotional dysregulation. EF correlates with adaptive and affective behaviors, highlighting the promise of behavioral interventions to target cognitive flexibility, emotional awareness, and reactivity in this population., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters, indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-Seq comparing osteoblasts differentiated from Ezh2R684C/+, and Ezh2+/+ BM-mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases KDM6A and KDM6B substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.

Individuals with Kabuki syndrome type 1 (KS1) often have hearing loss recognized in middle childhood. Current clinical dogma suggests that this phenotype is caused by frequent infections due to the immune deficiency in KS1 and/or secondary to structural abnormalities of the ear. To clarify some aspects of hearing loss, we collected information on hearing status from 21 individuals with KS1 and found that individuals have both sensorineural and conductive hearing loss, with the average age of presentation being 7 years. Our data suggest that while ear infections and structural abnormalities contribute to the observed hearing loss, these factors do not explain all loss. Using a KS1 mouse model, we found hearing abnormalities from hearing onset, as indicated by auditory brainstem response measurements. In contrast to mouse and human data for CHARGE syndrome, a disorder possessing overlapping clinical features with KS and a well-known cause of hearing loss and structural inner ear abnormalities, there are no apparent structural abnormalities of the cochlea in KS1 mice. The KS1 mice also display diminished distortion product otoacoustic emission levels, which suggests outer hair cell dysfunction. Combining these findings, our data suggests that KMT2D dysfunction causes sensorineural hearing loss compounded with external factors, such as infection.

The aim of this study was to provide a descriptive overview of the social characteristics associated with Wiedemann-Steiner syndrome (WSS). A total of 24 parents of children/adults with WSS (11F, mean age = 12.94 years, SD = 8.00) completed the Social Responsiveness Scale 2nd Edition (SRS-2); Colorado Learning Difficulties Questionnaire (CLDQ) and Strengths and Difficulties Questionnaire (SDQ). Almost half our sample reported a diagnosis of autism spectrum disorder (ASD) and 70% had intellectual disability. On the SDQ, over 90% of participants were rated in borderline/clinical ranges in Peer Problems, yet the majority fell within normal limits in Prosocial Behaviors. Most fell in the moderate/severe difficulties ranges across SRS-2 Social Cognition, Communication, and Restricted/Repetitive Behaviors scales (all >70%); whereas substantially less participants met these ranges for deficits in Social Awareness (50%) and Social Motivation (33.33%). A pattern of relatively strong prosocial skills and social drive in the context of difficulties with inflexible behaviors, social cognition, and communication was observed, regardless of gender, ASD or intellectual disability diagnosis. The social phenotype associated with WSS is characterized by some autistic features paired with unusually high social motivation and prosocial tendencies., (© 2023 Wiley Periodicals LLC.)

Objectives: Wiedemann-Steiner syndrome (WSS) is a rare Mendelian disorder of the epigenetic machinery caused by heterozygous pathogenic variants in KMT2A . Currently, the specific neurocognitive profile of this syndrome remains unknown. This case series provides insight into the cognitive phenotype of WSS., Methods: This study involves a retrospective medical chart review of 10 pediatric patients, each with a molecularly confirmed diagnosis of WSS who underwent clinical neuropsychological evaluation at an academic medical center., Results: The majority of patients performed in the below average to very low ranges in Nonverbal Reasoning, Visual/Spatial Perception, Visuoconstruction, Visual Memory, Attention, Working Memory and Math Computation skills. In contrast, over half the sample performed within normal limits on Receptive Vocabulary, Verbal Memory, and Word Reading. Wilcoxon signed rank test showed weaker Nonverbal versus Verbal Reasoning skills ( p  = .005). Most caregivers reported deficits in executive functioning, most notably in emotion regulation., Conclusions: Nonverbal reasoning/memory, visuospatial/construction, attention, working memory, executive functioning, and math computation skills are areas of weakness among those with WSS. These findings overlap with research on Kabuki syndrome, which is caused by variants in KMT2D, and suggest disruption in the neurogenesis of the hippocampal formation may drive shared pathogenesis of the two syndromes.

This study examined anxiety in Wiedemann-Steiner syndrome (WSS). Eighteen caregivers and participants with WSS completed the parent- and self-report versions of the Screen for Child Anxiety Related Disorder or the adapted version of the Screen for Adult Anxiety Related Disorder. Approximately 33.33% of parents and 65% of participants with WSS rated in the clinical range for overall anxiety. Across anxiety subtypes, parents primarily indicated concerns with Separation Anxiety (72%), which was also endorsed by the majority of participants with WSS (82%). The emergent trend showed Total Anxiety increased with age based on parent-informant ratings. The behavioral phenotype of WSS includes elevated anxiety. Clinical management should include incorporating early behavioral interventions to bolster emotion regulation given the observed risk of anxiety with age., (© 2022 Wiley Periodicals LLC.)

Background: Wiedemann-Steiner syndrome (WSS) is a rare genetic disorder caused by heterozygous variants in KMT2A. To date, the cognitive profile associated with WSS remains largely unknown, although emergent case series implicate increased risk of non-verbal reasoning and visual processing deficits. This study examines the academic and learning concerns associated with WSS based on a parent-report screening measure., Participants and Methods: A total of 25 parents of children/adults with a molecularly-confirmed diagnosis of WSS (mean age = 12.85 years, SD = 7.82) completed the Colorado Learning Difficulties Questionnaire (CLDQ), a parent-screening measure of learning and academic difficulties. Parent ratings were compared to those from a normative community sample to determine focal areas in Math, Reading and Spatial skills that may be weaker within this clinical population., Results: On average, parent ratings on the Math (mean Z = -3.08, SD = 0.87) and Spatial scales (mean Z = -2.52, SD = 0.85) were significantly more elevated than that of Reading (mean Z = -1.31, SD = 1.46) (Wilcoxon sign rank test Z < -3.83, P < 0.001), reflecting relatively more challenges observed in these areas. Distribution of parent ratings in Math items largely reflect a positively skewed distribution with most endorsing over three standard deviations below a community sample. In contrast, distributions of parent ratings in Reading and Spatial domains were more symmetric but flat. Ratings for Reading items yielded much larger variance than the other two domains, reflecting a wider range of performance variability., Conclusions: Parent ratings on the CLDQ suggest more difficulties with Math and Spatial skills among those with WSS within group and relative to a community sample. Study results are consistent with recent case reports on the neuropsychological profile associated with WSS and with Kabuki syndrome, which is caused by variants in the related gene KMT2D. Findings lend support for overlapping cognitive patterns across syndromes, implicating potential common disease pathogenesis., (© 2022 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)

An expanding range of genetic syndromes are characterized by genome-wide disruptions in DNA methylation profiles referred to as episignatures. Episignatures are distinct, highly sensitive, and specific biomarkers that have recently been applied in clinical diagnosis of genetic syndromes. Episignatures are contained within the broader disorder-specific genome-wide DNA methylation changes, which can share significant overlap among different conditions. In this study, we performed functional genomic assessment and comparison of disorder-specific and overlapping genome-wide DNA methylation changes related to 65 genetic syndromes with previously described episignatures. We demonstrate evidence of disorder-specific and recurring genome-wide differentially methylated probes (DMPs) and regions (DMRs). The overall distribution of DMPs and DMRs across the majority of the neurodevelopmental genetic syndromes analyzed showed substantial enrichment in gene promoters and CpG islands, and under-representation of the more variable intergenic regions. Analysis showed significant enrichment of the DMPs and DMRs in gene pathways and processes related to neurodevelopment, including neurogenesis, synaptic signaling and synaptic transmission. This study expands beyond the diagnostic utility of DNA methylation episignatures by demonstrating correlation between the function of the mutated genes and the consequent genomic DNA methylation profiles as a key functional element in the molecular etiology of genetic neurodevelopmental disorders., (© 2022 Wiley Periodicals LLC.)

Wiedemann-Steiner syndrome (WSS) is a rare genetic disorder caused by mutation in KMT2A and characterized by neurodevelopmental delay. This study is the first prospective investigation to examine the sleep and behavioral phenotypes among those with WSS through parent-informant screening inventories. A total of 24 parents of children/adults with WSS (11F, Mean age = 12.71 years, SD = 8.17) completed the Strengths and Difficulties Questionnaire (SDQ) and 22 of these caregivers also completed the Modified Simonds and Parraga Sleep Questionnaire (MSPSQ). On average, the majority of those with WSS (83%) were rated to show borderline to clinical level of behavioral difficulties on the SDQ. Approximately 83% were rated in these ranges for hyperactivity, 63% for emotional problems, and 50% for conduct problems. When applying prior published clinical cut-off for risk of sleep disturbance among those with neurodevelopmental disorders, over 80% of our sample exceeded this limit on the MSPSQ. Largely, caregivers' ratings suggested restless sleep, rigid bedtime rituals, sleep reluctance and breathing through the mouth in sleep were most consistent problems observed. Partial correlations between sleep and behavioral domains showed elevated emotional problems were associated with parasomnia characteristics after controlling for age. Daytime drowsiness and activity were associated with more hyperactivity. Those with more night waking problems and delayed sleep onset were rated to show more severe conduct problems. Overall, these findings suggest dysfunctional sleep behaviors, hyperactivity, and affective problems are part of the neurobehavioral phenotype of WSS. Routine clinical care for those affected by WSS should include close monitoring of sleep and overactive behaviors., Competing Interests: HB is a consultant for Mahzi therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DB declared a shared affiliation with the author JH to the handling editor at the time of review., (Copyright © 2022 Ng, Bjornsson, Fahrner and Harris.)

Sotos syndrome (SS), the most common overgrowth with intellectual disability (OGID) disorder, is caused by inactivating germline mutations of NSD1, which encodes a histone H3 lysine 36 methyltransferase. To understand how NSD1 inactivation deregulates transcription and DNA methylation (DNAm), and to explore how these abnormalities affect human development, we profiled transcription and DNAm in SS patients and healthy control individuals. We identified a transcriptional signature that distinguishes individuals with SS from controls and was also deregulated in NSD1-mutated cancers. Most abnormally expressed genes displayed reduced expression in SS; these downregulated genes consisted mostly of bivalent genes and were enriched for regulators of development and neural synapse function. DNA hypomethylation was strongly enriched within promoters of transcriptionally deregulated genes: overexpressed genes displayed hypomethylation at their transcription start sites while underexpressed genes featured hypomethylation at polycomb binding sites within their promoter CpG island shores. SS patients featured accelerated molecular aging at the levels of both transcription and DNAm. Overall, these findings indicate that NSD1-deposited H3K36 methylation regulates transcription by directing promoter DNA methylation, partially by repressing polycomb repressive complex 2 (PRC2) activity. These findings could explain the phenotypic similarity of SS to OGID disorders that are caused by mutations in PRC2 complex-encoding genes., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Overlapping clinical phenotypes and an expanding breadth and complexity of genomic associations are a growing challenge in the diagnosis and clinical management of Mendelian disorders. The functional consequences and clinical impacts of genomic variation may involve unique, disorder-specific, genomic DNA methylation episignatures. In this study, we describe 19 novel episignature disorders and compare the findings alongside 38 previously established episignatures for a total of 57 episignatures associated with 65 genetic syndromes. We demonstrate increasing resolution and specificity ranging from protein complex, gene, sub-gene, protein domain, and even single nucleotide-level Mendelian episignatures. We show the power of multiclass modeling to develop highly accurate and disease-specific diagnostic classifiers. This study significantly expands the number and spectrum of disorders with detectable DNA methylation episignatures, improves the clinical diagnostic capabilities through the resolution of unsolved cases and the reclassification of variants of unknown clinical significance, and provides further insight into the molecular etiology of Mendelian conditions., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

TET3 encodes an essential dioxygenase involved in epigenetic regulation through DNA demethylation. TET3 deficiency, or Beck-Fahrner syndrome (BEFAHRS; MIM: 618798), is a recently described neurodevelopmental disorder of the DNA demethylation machinery with a nonspecific phenotype resembling other chromatin-modifying disorders, but inconsistent variant types and inheritance patterns pose diagnostic challenges. Given TET3's direct role in regulating 5-methylcytosine and recent identification of syndrome-specific DNA methylation profiles, we analyzed genome-wide DNA methylation in whole blood of TET3-deficient individuals and identified an episignature that distinguishes affected and unaffected individuals and those with mono-allelic and bi-allelic pathogenic variants. Validation and testing of the episignature correctly categorized known TET3 variants and determined pathogenicity of variants of uncertain significance. Clinical utility was demonstrated when the episignature alone identified an affected individual from over 1000 undiagnosed cases and was confirmed upon distinguishing TET3-deficient individuals from those with 46 other disorders. The TET3-deficient signature - and the signature resulting from activating mutations in DNMT1 which normally opposes TET3 - are characterized by hypermethylation, which for BEFAHRS involves CpG sites that may be biologically relevant. This work expands the role of epi-phenotyping in molecular diagnosis and reveals genome-wide DNA methylation profiling as a quantitative, functional readout for characterization of this new biochemical category of disease., (© 2021. The Author(s).)

Wiedemann-Steiner syndrome (WSS) is an autosomal dominant disorder caused by monoallelic variants in KMT2A and characterized by intellectual disability and hypertrichosis. We performed a retrospective, multicenter, observational study of 104 individuals with WSS from five continents to characterize the clinical and molecular spectrum of WSS in diverse populations, to identify physical features that may be more prevalent in White versus Black Indigenous People of Color individuals, to delineate genotype-phenotype correlations, to define developmental milestones, to describe the syndrome through adulthood, and to examine clinicians' differential diagnoses. Sixty-nine of the 82 variants (84%) observed in the study were not previously reported in the literature. Common clinical features identified in the cohort included: developmental delay or intellectual disability (97%), constipation (63.8%), failure to thrive (67.7%), feeding difficulties (66.3%), hypertrichosis cubiti (57%), short stature (57.8%), and vertebral anomalies (46.9%). The median ages at walking and first words were 20 months and 18 months, respectively. Hypotonia was associated with loss of function (LoF) variants, and seizures were associated with non-LoF variants. This study identifies genotype-phenotype correlations as well as race-facial feature associations in an ethnically diverse cohort, and accurately defines developmental trajectories, medical comorbidities, and long-term outcomes in individuals with WSS., (© 2021 Wiley Periodicals LLC.)

Dubowitz syndrome (DubS) is considered a recognizable syndrome characterized by a distinctive facial appearance and deficits in growth and development. There have been over 200 individuals reported with Dubowitz or a "Dubowitz-like" condition, although no single gene has been implicated as responsible for its cause. We have performed exome (ES) or genome sequencing (GS) for 31 individuals clinically diagnosed with DubS. After genome-wide sequencing, rare variant filtering and computational and Mendelian genomic analyses, a presumptive molecular diagnosis was made in 13/27 (48%) families. The molecular diagnoses included biallelic variants in SKIV2L, SLC35C1, BRCA1, NSUN2; de novo variants in ARID1B, ARID1A, CREBBP, POGZ, TAF1, HDAC8, and copy-number variation at1p36.11(ARID1A), 8q22.2(VPS13B), Xp22, and Xq13(HDAC8). Variants of unknown significance in known disease genes, and also in genes of uncertain significance, were observed in 7/27 (26%) additional families. Only one gene, HDAC8, could explain the phenotype in more than one family (N = 2). All but two of the genomic diagnoses were for genes discovered, or for conditions recognized, since the introduction of next-generation sequencing. Overall, the DubS-like clinical phenotype is associated with extensive locus heterogeneity and the molecular diagnoses made are for emerging clinical conditions sharing characteristic features that overlap the DubS phenotype., (© 2020 Wiley Periodicals LLC.)

Background: Ultrarare Marshall-Smith and Malan syndromes, caused by changes of the gene nuclear factor I X (NFIX), are characterised by intellectual disability (ID) and behavioural problems, although questions remain. Here, development and behaviour are studied and compared in a cross-sectional study, and results are presented with genetic findings., Methods: Behavioural phenotypes are compared of eight individuals with Marshall-Smith syndrome (three male individuals) and seven with Malan syndrome (four male individuals). Long-term follow-up assessment of cognition and adaptive behaviour was possible in three individuals with Marshall-Smith syndrome., Results: Marshall-Smith syndrome individuals have more severe ID, less adaptive behaviour, more impaired speech and less reciprocal interaction compared with individuals with Malan syndrome. Sensory processing difficulties occur in both syndromes. Follow-up measurement of cognition and adaptive behaviour in Marshall-Smith syndrome shows different individual learning curves over time., Conclusions: Results show significant between and within syndrome variability. Different NFIX variants underlie distinct clinical phenotypes leading to separate entities. Cognitive, adaptive and sensory impairments are common in both syndromes and increase the risk of challenging behaviour. This study highlights the value of considering behaviour within developmental and environmental context. To improve quality of life, adaptations to environment and treatment are suggested to create a better person-environment fit., (© 2020 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)

Purpose: Tatton-Brown-Rahman syndrome (TBRS) is a newly defined genetic entity characterized by overgrowth and intellectual disability, resulting from germline mutations in the gene encoding DNA methyltransferase 3 alpha (DNMT3A). Affected individuals with benign and malignant tumors have been reported; to our knowledge pituitary adenomas (and other tumors identified in our patient) have not yet been described in this syndrome., Case: We report the case of a 34-year-old woman with TBRS who developed a GH-secreting pituitary macroadenoma and other benign tumors and cystic lesions involving diverse organ systems. Whole-exome sequencing revealed a heterozygous, likely pathogenic variant (c.700&#95;709 del10, p. Gly234ArgfsX79) in exon7 of DNMT3A, and a heterozygous variant of uncertain significance (c.25 C>T, p.Arg9Trp) in exon 1 of the gene encoding aryl hydrocarbon receptor-interacting protein (AIP). The patient failed somatostatin analog treatment, and underwent surgery. The tumor retained AIP expression, and analysis of tumor DNA indicated the presence of both AIP alleles, consistent with no loss of heterozygosity. These findings suggest that the AIP variant was not the primary driver of pituitary adenoma development., Conclusion: Our case suggests that TBRS might be associated with pituitary adenoma and a broader spectrum of tumors than previously thought, making long-term follow up of these patients crucial to identify tumors early, and to elucidate the clinical spectrum of the disorder for optimization of management.

Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation (5-methylcytosine [5mC]) of DNA is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has yet been delineated. Here, we describe in detail a Mendelian disorder caused by the disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. We identify and characterize 11 cases of human TET3 deficiency in eight families with the common phenotypic features of intellectual disability and/or global developmental delay; hypotonia; autistic traits; movement disorders; growth abnormalities; and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues; all but one such variant occur within the catalytic domain, and most display hypomorphic function in an assay of catalytic activity. TET3 deficiency and other Mendelian disorders of the epigenetic machinery show substantial phenotypic overlap, including features of intellectual disability and abnormal growth, underscoring shared disease mechanisms., (Copyright © 2019 American Society of Human Genetics. All rights reserved.)

The epigenetic machinery in conjunction with the transcriptional machinery is responsible for maintaining genome-wide chromatin states and dynamically regulating gene expression. Mendelian disorders of the epigenetic machinery (MDEMs) are genetic disorders resulting from mutations in components of the epigenetic apparatus. Though individually rare, MDEMs have emerged as a collectively common etiology for intellectual disability (ID) and growth disruption. Studies in model organisms and humans have demonstrated dosage sensitivity of this gene group with haploinsufficiency as a predominant disease mechanism. The epigenetic machinery consists of three enzymatic components (writers, erasers and chromatin remodelers) as well as one non-enzymatic group (readers). A tally of the entire census of such factors revealed that although multiple enzymatic activities never coexist within a single component, individual enzymatic activities often coexist with a reader domain. This group of disorders disrupts both the chromatin and transcription states of target genes downstream of the given component but also DNA methylation on a global scale. Elucidation of these global epigenetic changes may inform our understanding of disease pathogenesis and have diagnostic utility. Moreover, many therapies targeting epigenetic marks already exist, and some have proven successful in treating cancer. This, along with the recent observation that neurological dysfunction in these disorders may in fact be treatable in postnatal life, suggests that the scientific community should prioritize this group as a potentially treatable cause of ID. Here we summarize the recent expansion and major characteristics of MDEMs, as well as the unique therapeutic prospects for this group of disorders., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Kabuki 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.

Purpose of Review: Sotos syndrome is among a growing list of disorders resulting from mutations in epigenetic machinery genes. These Mendelian disorders of the epigenetic machinery (MDEMs) exhibit phenotypic overlap broadly characterized by intellectual disability and atypical growth and behaviors. Manifestations of Sotos syndrome include a distinct facial appearance, overgrowth, intellectual disability, and behavioral issues. Herein we review key aspects of Sotos syndrome, focusing on the neurobehavioral phenotype. Additionally, we highlight recent advances in our understanding of molecular pathogenesis implicating epigenetic mechanisms., Recent Findings: Increasing evidence suggests MDEMs account for ∼19% of intellectual disability and ∼45% of overgrowth combined with intellectual disability, with Sotos syndrome constituting most of the latter. Although the genetic cause of Sotos syndrome, disruption of the histone methyltransferase writer NSD1, is well established, recent studies have further delineated the neurobehavioral phenotype and provided insight into disease pathogenesis. Explicitly, NSD1 target genes accounting for a subset of Sotos syndrome features and a specific DNA methylation signature have been identified., Summary: Sotos syndrome is, therefore, a genetic disorder with epigenetic consequences. Its characteristic neurobehavioral phenotype and those of related MDEMs illustrate the essential role epigenetic mechanisms play in neurologic development. Improvement in our understanding of molecular pathogenesis has important implications for development of diagnostic tests and therapeutic interventions.

Background: Deleterious mutations in cytosolic leucine-tRNA synthetase (LARS) cause infantile liver failure syndrome, type 1 (ILFS1), a recently recognized, rare autosomal recessive disorder (OMIM151350). Only six families with ILFS1 have been reported in the literature. Patients with ILFS1 are typically diagnosed between 5 and 24 months of age with failure to thrive, developmental delays, encephalopathy, microcytic anemia, and chronic liver dysfunction with recurrent exacerbations following childhood illnesses. Neonatal manifestations of this disorder have not been well documented., Case Report: We report a premature female newborn with intrauterine growth restriction, failure to thrive, congenital anemia, anasarca, and fulminant liver failure leading to lethal multiple organ failure. Liver failure in this infant was characterized by a disproportionate impairment of liver synthetic function, including severe coagulopathy and hypoalbuminemia without significant defects in liver detoxification or evidence of hepatocellular injury during early phase of the disease. Whole-exome sequencing of child-parent trio identified two inherited missense mutations in LARS in this patient. One, c.1292T>A; p.Val431Asp, has been reported in patients with ILFS1, while the other, c.725C>T; p.Pro242Leu, is novel. Both mutations involve amino acid residues in the highly conserved editing domain of LARS, are predicted to be functionally deleterious, and presumably contribute to the clinical manifestations in this patient., Conclusion: This is the first case documenting neonatal manifestation of ILFS1, highlighting early, severe, and disproportionate defects in liver synthetic function. Timely diagnosis of ILFS1 is crucial to guide critical clinical management and improve outcomes of this rare and potentially life-threatening disorder.

Malan syndrome is an overgrowth disorder described in a limited number of individuals. We aim to delineate the entity by studying a large group of affected individuals. We gathered data on 45 affected individuals with a molecularly confirmed diagnosis through an international collaboration and compared data to the 35 previously reported individuals. Results indicate that height is > 2 SDS in infancy and childhood but in only half of affected adults. Cardinal facial characteristics include long, triangular face, macrocephaly, prominent forehead, everted lower lip, and prominent chin. Intellectual disability is universally present, behaviorally anxiety is characteristic. Malan syndrome is caused by deletions or point mutations of NFIX clustered mostly in exon 2. There is no genotype-phenotype correlation except for an increased risk for epilepsy with 19p13.2 microdeletions. Variants arose de novo, except in one family in which mother was mosaic. Variants causing Malan and Marshall-Smith syndrome can be discerned by differences in the site of stop codon formation. We conclude that Malan syndrome has a well recognizable phenotype that usually can be discerned easily from Marshall-Smith syndrome but rarely there is some overlap. Differentiation from Sotos and Weaver syndrome can be made by clinical evaluation only., (© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

DNM1L encodes dynamin-related protein 1 (DRP1/DLP1), a key component of the mitochondrial fission machinery that is essential for proper functioning of the mammalian brain. Previously reported probands with de novo missense mutations in DNM1L presented in the first year of life with severe encephalopathy and refractory epilepsy, with several dying within the first several weeks after birth. In contrast, we report identical novel missense mutations in DNM1L in two unrelated probands who experienced normal development for several years before presenting with refractory focal status epilepticus and subsequent rapid neurological decline. We expand the phenotype of DNM1L-related mitochondrial fission defects, reveal common unique clinical characteristics and imaging findings, and compare the cellular impact of this novel mutation to the previously reported A395D lethal variant. We demonstrate that our R403C mutation, which resides in the assembly region of DRP1, acts by a dominant-negative mechanism and reduces oligomerization, mitochondrial fission activity, and mitochondrial recruitment of DRP1, but to a lesser extent compared to the A395D mutation. In contrast to the initial report of neonatal lethality resulting from DNM1L mutation and DRP1 dysfunction, our results show that milder DRP1 impairment is compatible with normal early development and subsequently results in a distinct set of neurological findings. In addition, we identify a common pathogenic mechanism whereby DNM1L mutations impair mitochondrial fission. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

DNA sequencing has become a powerful method to discover the genetic basis of disease. Standard, widely used protocols for analysis usually begin by comparing each individual to the human reference genome. When applied to a set of related individuals, this approach reveals millions of differences, most of which are shared among the individuals and unrelated to the disease being investigated. We have developed a novel algorithm for variant detection, one that compares DNA sequences directly to one another, without aligning them to the reference genome. When used to find de novo mutations in exome sequences from family trios, or to compare normal and diseased samples from the same individual, the new method, direct alignment for mutation discovery (DIAMUND), produces a dramatically smaller list of candidate mutations than previous methods, without losing sensitivity to detect the true cause of a genetic disease. We demonstrate our results on several example cases, including two family trios in which it correctly found the disease-causing variant while excluding thousands of harmless variants that standard methods had identified., (© 2013 The Authors. *Human Mutation published by Wiley Periodicals, Inc.)

Mendelian disorders of the epigenetic machinery are a newly delineated group of multiple congenital anomaly and intellectual disability syndromes resulting from mutations in genes encoding components of the epigenetic machinery. The gene products affected in these inherited conditions act in trans and are expected to have widespread epigenetic consequences. Many of these syndromes demonstrate phenotypic overlap with classical imprinting disorders and with one another. The various writer and eraser systems involve opposing players, which we propose must maintain a balance between open and closed chromatin states in any given cell. An imbalance might lead to disrupted expression of disease-relevant target genes. We suggest that classifying disorders based on predicted effects on this balance would be informative regarding pathogenesis. Furthermore, strategies targeted at restoring this balance might offer novel therapeutic avenues, taking advantage of available agents such as histone deacetylase inhibitors and histone acetylation antagonists.

Adenylosuccinate lyase (ADSL) deficiency is a rare inborn error of metabolism resulting in accumulation of metabolites including succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (S-Ado) in the brain and other tissues. Patients with ADSL have progressive psychomotor retardation, neonatal seizures, global developmental delay, hypotonia, and autistic features, although variable clinical manifestations may make the initial diagnosis challenging. Two cases of the severe form of the disease are reported here: an 18-month-old boy with global developmental delay, intractable neonatal seizures, progressive cerebral atrophy, and marked hypomyelination, and a 3-month-old girl presenting with microcephaly, neonatal seizures, and marked psychomotor retardation. In both patients in vivo proton magnetic resonance spectroscopy (MRS) showed the presence of S-Ado signal at 8.3 ppm, consistent with a prior report. Interestingly, SAICAr signal was also detectable at 7.5 ppm in affected white matter, which has not been reported in vivo before. A novel splice-site mutation, c.IVS12 + 1/G > C, in the ADSL gene was identified in the second patient. Our findings confirm the utility of in vivo proton MRS in suggesting a specific diagnosis of ADSL deficiency, and also demonstrate an additional in vivo resonance (7.5 ppm) of SAICAr in the cases of severe disease., (Copyright © 2012 Wiley Periodicals, Inc.)

The RAS-MAPK pathway is critical for human growth and development. Abnormalities at different steps of this signaling cascade result in neuro-cardio-facial-cutaneous syndromes, or the RASopathies, a group of disorders with overlapping yet distinct phenotypes. RASopathy patients have variable degrees of intellectual disability, poor growth, relative macrocephaly, ectodermal abnormalities, dysmorphic features, and increased risk for certain malignancies. Congenital heart disease, particularly hypertrophic cardiomyopathy (HCM) and pulmonic stenosis, are prominent features in these disorders. Significant locus heterogeneity exists for many of the RASopathies. Traditionally, these diseases were thought to be inherited in an autosomal dominant manner. However, recently patients with defects in two components of this pathway and overlapping features of various forms of Noonan syndrome and neurofibromatosis 1 and have been reported. Here we present a patient with severe, progressive neonatal HCM, elevated urinary catecholamine metabolites, and dysmorphic features in whom we identified a known LEOPARD syndrome-associated PTPN11 mutation (c.1403 C > T; p.T468M) and a novel, potentially pathogenic missense SOS1 variant (c.1018 C > T; p.P340S) replacing a rigid nonpolar imino acid with a polar amino acid at a highly conserved position. We describe detailed clinical manifestations, cardiac histopathology, and the molecular genetic findings. Oligogenic models of inheritance with potential synergistic effects should be considered in the RASopathies., (Copyright © 2012 Wiley Periodicals, Inc.)

Recent work suggests a link between the polycomb group protein EZH2 and mediation of gene silencing in association with maintenance of DNA methylation. However, we show that whereas basally expressed target cancer genes with minimal DNA methylation have increased transcription during EZH2 knockdown, densely DNA hypermethylated and silenced genes retain their methylation and remain transcriptionally silent. These results suggest that EZH2 can modulate transcription of basally expressed genes but not silent genes that are densely DNA methylated.

Histone H3 lysine 9 (H3K9) and lysine 27 (H3K27) trimethylation are properties of stably silenced heterochromatin whereas H3K9 dimethylation (H3K9me2) is important for euchromatic gene repression. In colorectal cancer cells, all of these marks, as well as the key enzymes which establish them, surround the hMLH1 promoter when it is DNA hypermethylated and aberrantly silenced, but are absent when the gene is unmethylated and fully expressed in a euchromatic state. When the aberrantly silenced gene is DNA demethylated and reexpressed following 5-aza-2'-deoxycytidine treatment, H3K9me1 and H3K9me2 are the only silencing marks that are lost. A series of other silenced and DNA hypermethylated gene promoters behave identically even when the genes are chronically DNA demethylated and reexpressed after genetic knockout of DNA methyltransferases. Our data indicate that when transcription of DNA hypermethylated genes is activated in cancer cells, their promoters remain in an environment with certain heterochromatic characteristics. This finding has important implications for the translational goal of reactivating aberrantly silenced cancer genes as a therapeutic maneuver.

We examined the relationship between aberrant DNA hypermethylation and key histone code components at a hypermethylated, silenced tumor suppressor gene promoter in human cancer. In lower eukaryotes, methylated H3-lysine 9 (methyl-H3-K9) determines DNA methylation and correlates with repressed gene transcription. Here we show that a zone of deacetylated histone H3 plus methyl-H3-K9 surrounds a hypermethylated, silenced hMLH1 promoter, which, when unmethylated and active, is embedded in methyl-H3-K4 and acetylated H3. Inhibiting DNA methyltransferases, but not histone deacetylases, leads first to promoter demethylation, second to gene reexpression, and finally to complete histone code reversal. Our findings suggest a new paradigm-DNA methylation may directly, or indirectly by inhibiting transcription, maintain key repressive elements of the histone code at a hypermethylated gene promoter in cancer.

Lumenal delivery of adenovirus vectors (AdV) results in inefficient gene transfer to human airway epithelium. The human coxsackievirus and adenovirus receptor (hCAR) was detected by immunofluorescence selectively at the basolateral surfaces of freshly excised human airway epithelial cells, suggesting that the absence of apical hCAR constitutes a barrier to adenovirus-mediated gene delivery in vivo. In transfected polarized Madin-Darby canine kidney cells, wild-type hCAR was expressed selectively at the basolateral membrane, whereas hCAR lacking the transmembrane and/or cytoplasmic domains was expressed on both the basolateral and apical membranes. Cells expressing apical hCAR still were not efficiently transduced by AdV applied to the apical surface. However, after the cells were treated with agents that remove components of the apical surface glycocalyx, AdV transduction occurred. These results indicate that adenovirus can infect via receptors located at the apical cell membrane but that the glycocalyx impedes interaction of AdV with apical receptors.