Monoallelic de novo AJAP1 loss-of-function variants disrupt trans-synaptic control of neurotransmitter release.

Adherens junction-associated protein 1 (AJAP1) has been implicated in brain diseases; however, a pathogenic mechanism has not been identified. AJAP1 is widely expressed in neurons and binds to γ-aminobutyric acid type B receptors (GBRs), which inhibit neurotransmitter release at most synapses in the brain. Here, we show that AJAP1 is selectively expressed in dendrites and trans-synaptically recruits GBRs to presynaptic sites of neurons expressing AJAP1. We have identified several monoallelic AJAP1 variants in individuals with epilepsy and/or neurodevelopmental disorders. Specifically, we show that the variant p.(W183C) lacks binding to GBRs, resulting in the inability to recruit them. Ultrastructural analysis revealed significantly decreased presynaptic GBR levels in Ajap1-/- and Ajap1W183C/+ mice. Consequently, these mice exhibited reduced GBR-mediated presynaptic inhibition at excitatory and inhibitory synapses, along with impaired synaptic plasticity. Our study reveals that AJAP1 enables the postsynaptic neuron to regulate the level of presynaptic GBR-mediated inhibition, supporting the clinical relevance of loss-of-function AJAP1 variants.

Comprehensive EHMT1 variants analysis broadens genotype-phenotype associations and molecular mechanisms in Kleefstra syndrome.

The shift to a genotype-first approach in genetic diagnostics has revolutionized our understanding of neurodevelopmental disorders, expanding both their molecular and phenotypic spectra. Kleefstra syndrome (KLEFS1) is caused by EHMT1 haploinsufficiency and exhibits broad clinical manifestations. EHMT1 encodes euchromatic histone methyltransferase-1-a pivotal component of the epigenetic machinery. We have recruited 209 individuals with a rare EHMT1 variant and performed comprehensive molecular in silico and in vitro testing alongside DNA methylation (DNAm) signature analysis for the identified variants. We (re)classified the variants as likely pathogenic/pathogenic (molecularly confirming Kleefstra syndrome) in 191 individuals. We provide an updated and broader clinical and molecular spectrum of Kleefstra syndrome, including individuals with normal intelligence and familial occurrence. Analysis of the EHMT1 variants reveals a broad range of molecular effects and their associated phenotypes, including distinct genotype-phenotype associations. Notably, we showed that disruption of the "reader" function of the ankyrin repeat domain by a protein altering variant (PAV) results in a KLEFS1-specific DNAm signature and milder phenotype, while disruption of only "writer" methyltransferase activity of the SET domain does not result in KLEFS1 DNAm signature or typical KLEFS1 phenotype. Similarly, N-terminal truncating variants result in a mild phenotype without the DNAm signature. We demonstrate how comprehensive variant analysis can provide insights into pathogenesis of the disorder and DNAm signature. In summary, this study presents a comprehensive overview of KLEFS1 and EHMT1, revealing its broader spectrum and deepening our understanding of its molecular mechanisms, thereby informing accurate variant interpretation, counseling, and clinical management.

Prevalence of comorbidities in individuals with neurodevelopmental disorders from the aggregated phenomics data of 51,227 pediatric individuals.

The prevalence of comorbidities in individuals with neurodevelopmental disorders (NDDs) is not well understood, yet these are important for accurate diagnosis and prognosis in routine care and for characterizing the clinical spectrum of NDD syndromes. We thus developed PhenomAD-NDD, an aggregated database containing the comorbid phenotypic data of 51,227 individuals with NDD, all harmonized into Human Phenotype Ontology (HPO), with in total 3,054 unique HPO terms. We demonstrate that almost all congenital anomalies are more prevalent in the NDD population than in the general population, and the NDD baseline prevalence allows for an approximation of the enrichment of symptoms. For example, such analyses of 33 genetic NDDs show that 32% of enriched phenotypes are currently not reported in the clinical synopsis in the Online Mendelian Inheritance in Man (OMIM). PhenomAD-NDD is open to all via a visualization online tool and allows us to determine the enrichment of symptoms in NDD.

DNA methylation episignature and comparative epigenomic profiling for Pitt-Hopkins syndrome caused by TCF4 variants.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by pathogenic variants in TCF4, leading to intellectual disability, specific morphological features, and autonomic nervous system dysfunction. Epigenetic dysregulation has been implicated in PTHS, prompting the investigation of a DNA methylation (DNAm) "episignature" specific to PTHS for diagnostic purposes and variant reclassification and functional insights into the molecular pathophysiology of this disorder. A cohort of 67 individuals with genetically confirmed PTHS and three individuals with intellectual disability and a variant of uncertain significance (VUS) in TCF4 were studied. The DNAm episignature was developed with an Infinium Methylation EPIC BeadChip array analysis using peripheral blood cells. Support vector machine (SVM) modeling and clustering methods were employed to generate a DNAm classifier for PTHS. Validation was extended to an additional cohort of 11 individuals with PTHS. The episignature was assessed in relation to other neurodevelopmental disorders and its specificity was examined. A specific DNAm episignature for PTHS was established. The classifier exhibited high sensitivity for TCF4 haploinsufficiency and missense variants in the basic-helix-loop-helix domain. Notably, seven individuals with TCF4 variants exhibited negative episignatures, suggesting complexities related to mosaicism, genetic factors, and environmental influences. The episignature displayed degrees of overlap with other related disorders and biological pathways. This study defines a DNAm episignature for TCF4-related PTHS, enabling improved diagnostic accuracy and VUS reclassification. The finding that some cases scored negatively underscores the potential for multiple or nested episignatures and emphasizes the need for continued investigation to enhance specificity and coverage across PTHS-related variants.

Further delineation of the rare GDACCF (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies syndrome): genotype and phenotype of 22 patients with ZNF148 mutations.

BACKGROUND: Pathogenic variants in the zinc finger protein coding genes are rare causes of intellectual disability and congenital malformations. Mutations in the ZNF148 gene causing GDACCF syndrome (global developmental delay, absent or hypoplastic corpus callosum, dysmorphic facies; MIM #617260) have been reported in five individuals so far. METHODS: As a result of an international collaboration using GeneMatcher Phenome Central Repository and personal communications, here we describe the clinical and molecular genetic characteristics of 22 previously unreported individuals. RESULTS: The core clinical phenotype is characterised by developmental delay particularly in the domain of speech development, postnatal growth retardation, microcephaly and facial dysmorphism. Corpus callosum abnormalities appear less frequently than suggested by previous observations. The identified mutations concerned nonsense or frameshift variants that were mainly located in the last exon of the ZNF148 gene. Heterozygous deletion including the entire ZNF148 gene was found in only one case. Most mutations occurred de novo, but were inherited from an affected parent in two families. CONCLUSION: The GDACCF syndrome is clinically diverse, and a genotype-first approach, that is, exome sequencing is recommended for establishing a genetic diagnosis rather than a phenotype-first approach. However, the syndrome may be suspected based on some recurrent, recognisable features. Corpus callosum anomalies were not as constant as previously suggested, we therefore recommend to replace the term 'GDACCF syndrome' with 'ZNF148-related neurodevelopmental disorder'.

PhenoScore quantifies phenotypic variation for rare genetic diseases by combining facial analysis with other clinical features using a machine-learning framework.

Several molecular and phenotypic algorithms exist that establish genotype-phenotype correlations, including facial recognition tools. However, no unified framework that investigates both facial data and other phenotypic data directly from individuals exists. We developed PhenoScore: an open-source, artificial intelligence-based phenomics framework, combining facial recognition technology with Human Phenotype Ontology data analysis to quantify phenotypic similarity. Here we show PhenoScore's ability to recognize distinct phenotypic entities by establishing recognizable phenotypes for 37 of 40 investigated syndromes against clinical features observed in individuals with other neurodevelopmental disorders and show it is an improvement on existing approaches. PhenoScore provides predictions for individuals with variants of unknown significance and enables sophisticated genotype-phenotype studies by testing hypotheses on possible phenotypic (sub)groups. PhenoScore confirmed previously known phenotypic subgroups caused by variants in the same gene for SATB1, SETBP1 and DEAF1 and provides objective clinical evidence for two distinct ADNP-related phenotypes, already established functionally.

Genetic diagnostic approach to intellectual disability and multiple congenital anomalies in Indonesia.

Intellectual disability (ID) and multiple congenital anomalies (MCA) are major contributors to infant mortality, childhood morbidity, and long-term disability, with multifactorial aetiology including genetics. We aim to set a diagnostic approach for genetic evaluation of patients with ID and MCA, which can be applied efficiently with a good diagnostic rate in Indonesia or other low resources settings. Out of 131 ID cases, twenty-three individuals with ID/global developmental delay (GDD) and MCA were selected from two-steps of dysmorphology screening and evaluation. Genetic analysis included chromosomal microarray (CMA) analysis, targeted panel gene sequencing, and exome sequencing (ES). CMA revealed conclusive results for seven individuals. Meanwhile, two out of four cases were diagnosed by targeted gene sequencing. Five out of seven individuals were diagnosed using ES testing. Based on the experience, a novel and comprehensive flowchart combining thorough physical and dysmorphology evaluation, followed by suitable genetic tests is proposed as a diagnostic approach to elucidate the genetic factor(s) of ID/GDD and MCA in low resources settings such as Indonesia.

CERT1 mutations perturb human development by disrupting sphingolipid homeostasis.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Diagnosing, discarding, or de-VUSsing: A practical guide to (un)targeted metabolomics as variant-transcending functional tests.

PURPOSE: For patients with inherited metabolic disorders (IMDs), any diagnostic delay should be avoided because early initiation of personalized treatment could prevent irreversible health damage. To improve diagnostic interpretation of genetic data, gene function tests can be valuable assets. For IMDs, variant-transcending functional tests are readily available through (un)targeted metabolomics assays. To support the application of metabolomics for this purpose, we developed a gene-based guide to select functional tests to either confirm or exclude an IMD diagnosis. METHODS: Using information from a diagnostic IMD exome panel, Kyoto Encyclopedia of Genes and Genomes, and Inborn Errors of Metabolism Knowledgebase, we compiled a guide for metabolomics-based gene function tests. From our practical experience with this guide, we retrospectively selected illustrative cases for whom combined metabolomic/genomic testing improved diagnostic success and evaluated the effect hereof on clinical management. RESULTS: The guide contains 2047 metabolism-associated genes for which a validated or putative variant-transcending gene function test is available. We present 16 patients for whom metabolomic testing either confirmed or ruled out the presence of a second pathogenic variant, validated or ruled out pathogenicity of variants of uncertain significance, or identified a diagnosis initially missed by genetic analysis. CONCLUSION: Metabolomics-based gene function tests provide additional value in the diagnostic trajectory of patients with suspected IMD by enhancing and accelerating diagnostic success.

The MAP3K7 gene: Further delineation of clinical characteristics and genotype/phenotype correlations.

Mitogen-activated protein 3 kinase 7 (MAP3K7) encodes the ubiquitously expressed transforming growth factor ß-activated kinase 1, which plays a crucial role in many cellular processes. Mutationsin the MAP3K7 gene have been linked to two distinct disorders: frontometaphyseal dysplasia type 2 (FMD2) and cardiospondylocarpofacial syndrome (CSCF). The fact that different mutations can induce two distinct phenotypes suggests a phenotype/genotype correlation, but no side-by-side comparison has been done thus far to confirm this. Here, we significantly expand the cohort and the description of clinical phenotypes for patients with CSCF and FMD2 who carry mutations in MAP3K7. Our findings support that in contrast to FMD2-causing mutations, CSCF-causing mutations in MAP3K7 have a loss-of-function effect. Additionally, patients with pathogenic mutations in MAP3K7 are at risk for (severe) cardiac disease, have symptoms associated with connective tissue disease, and we show overlap in clinical phenotypes of CSCF with Noonan syndrome (NS). Together, we confirm a molecular fingerprint of FMD2- versus CSCF-causing MAP3K7 mutations and conclude that mutations in MAP3K7 should be considered in the differential diagnosis of patients with syndromic congenital cardiac defects and/or cardiomyopathy, syndromic connective tissue disorders, and in the differential diagnosis of NS.

Social motivation a relative strength in DYRK1A syndrome on a background of significant speech and language impairments.

Speech and language impairments are commonly reported in DYRK1A syndrome. Yet, speech and language abilities have not been systematically examined in a prospective cohort study. Speech, language, social behaviour, feeding, and non-verbal communication skills were assessed using standardised tools. The broader health and medical phenotype was documented using caregiver questionnaires, interviews and confirmation with medical records. 38 individuals with DYRK1A syndrome (23 male, median age 8 years 3 months, range 1 year 7 months to 25 years) were recruited. Moderate to severe intellectual disability (ID), autism spectrum disorder (ASD), vision, motor and feeding impairments were common, alongside epilepsy in a third of cases. Speech and language was disordered in all participants. Many acquired some degree of verbal communication, yet few (8/38) developed sufficient oral language skills to rely solely on verbal communication. Speech was characterised by severe apraxia and dysarthria in verbal participants, resulting in markedly poor intelligibility. Those with limited verbal language (30/38) used a combination of sign and graphic augmentative and alternative communication (AAC) systems. Language skills were low across expressive, receptive, and written domains. Most had impaired social behaviours (25/29). Restricted and repetitive interests were most impaired, whilst social motivation was a relative strength. Few individuals with DYRK1A syndrome use verbal speech as their sole means of communication, and hence, all individuals need early access to tailored, graphic AAC systems to support their communication. For those who develop verbal speech, targeted therapy for apraxia and dysarthria should be considered to improve intelligibility and, consequently, communication autonomy.

Phenotype based prediction of exome sequencing outcome using machine learning for neurodevelopmental disorders.

PURPOSE: Although the introduction of exome sequencing (ES) has led to the diagnosis of a significant portion of patients with neurodevelopmental disorders (NDDs), the diagnostic yield in actual clinical practice has remained stable at approximately 30%. We hypothesized that improving the selection of patients to test on the basis of their phenotypic presentation will increase diagnostic yield and therefore reduce unnecessary genetic testing. METHODS: We tested 4 machine learning methods and developed PredWES from these: a statistical model predicting the probability of a positive ES result solely on the basis of the phenotype of the patient. RESULTS: We first trained the tool on 1663 patients with NDDs and subsequently showed that diagnostic ES on the top 10% of patients with the highest probability of a positive ES result would provide a diagnostic yield of 56%, leading to a notable 114% increase. Inspection of our model revealed that for patients with NDDs, comorbid abnormal (lower) muscle tone and microcephaly positively correlated with a conclusive ES diagnosis, whereas autism was negatively associated with a molecular diagnosis. CONCLUSION: In conclusion, PredWES allows prioritizing patients with NDDs eligible for diagnostic ES on the basis of their phenotypic presentation to increase the diagnostic yield, making a more efficient use of health care resources.

Clustered mutations in the GRIK2 kainate receptor subunit gene underlie diverse neurodevelopmental disorders.

Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.

A Case Series of Familial ARID1B Variants Illustrating Variable Expression and Suggestions to Update the ACMG Criteria.

ARID1B is one of the most frequently mutated genes in intellectual disability (~1%). Most variants are readily classified, since they are de novo and are predicted to lead to loss of function, and therefore classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines for the interpretation of sequence variants. However, familial loss-of-function variants can also occur and can be challenging to interpret. Such variants may be pathogenic with variable expression, causing only a mild phenotype in a parent. Alternatively, since some regions of the ARID1B gene seem to be lacking pathogenic variants, loss-of-function variants in those regions may not lead to ARID1B haploinsufficiency and may therefore be benign. We describe 12 families with potential loss-of-function variants, which were either familial or with unknown inheritance and were in regions where pathogenic variants have not been described or are otherwise challenging to interpret. We performed detailed clinical and DNA methylation studies, which allowed us to confidently classify most variants. In five families we observed transmission of pathogenic variants, confirming their highly variable expression. Our findings provide further evidence for an alternative translational start site and we suggest updates for the ACMG guidelines for the interpretation of sequence variants to incorporate DNA methylation studies and facial analyses.

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology.

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.

Clinical delineation of SETBP1 haploinsufficiency disorder.

SETBP1 haploinsufficiency disorder (MIM#616078) is caused by haploinsufficiency of SETBP1 on chromosome 18q12.3, but there has not yet been any systematic evaluation of the major features of this monogenic syndrome, assessing penetrance and expressivity. We describe the first comprehensive study to delineate the associated clinical phenotype, with findings from 34 individuals, including 24 novel cases, all of whom have a SETBP1 loss-of-function variant or single (coding) gene deletion, confirmed by molecular diagnostics. The most commonly reported clinical features included mild motor developmental delay, speech impairment, intellectual disability, hypotonia, vision impairment, attention/concentration deficits, and hyperactivity. Although there is a mild overlap in certain facial features, the disorder does not lead to a distinctive recognizable facial gestalt. As well as providing insight into the clinical spectrum of SETBP1 haploinsufficiency disorder, this reports puts forward care recommendations for patient management.

Speech and language deficits are central to SETBP1 haploinsufficiency disorder.

Expressive communication impairment is associated with haploinsufficiency of SETBP1, as reported in small case series. Heterozygous pathogenic loss-of-function (LoF) variants in SETBP1 have also been identified in independent cohorts ascertained for childhood apraxia of speech (CAS), warranting further investigation of the roles of this gene in speech development. Thirty-one participants (12 males, aged 0; 8-23; 2 years, 28 with pathogenic SETBP1 LoF variants, 3 with 18q12.3 deletions) were assessed for speech, language and literacy abilities. Broader development was examined with standardised motor, social and daily life skills assessments. Gross and fine motor deficits (94%) and intellectual impairments (68%) were common. Protracted and aberrant speech development was consistently seen, regardless of motor or intellectual ability. We expand the linguistic phenotype associated with SETBP1 LoF syndrome (SETBP1 haploinsufficiency disorder), revealing a striking speech presentation that implicates both motor (CAS, dysarthria) and language (phonological errors) systems, with CAS (80%) being the most common diagnosis. In contrast to past reports, the understanding of language was rarely better preserved than language expression (29%). Language was typically low, to moderately impaired, with commensurate expression and comprehension ability. Children were sociable with a strong desire to communicate. Minimally verbal children (32%) augmented speech with sign language, gestures or digital devices. Overall, relative to general development, spoken language and literacy were poorer than social, daily living, motor and adaptive behaviour skills. Our findings show that poor communication is a central feature of SETBP1 haploinsufficiency disorder, confirming this gene as a strong candidate for speech and language disorders.

Disruptive mutations in TANC2 define a neurodevelopmental syndrome associated with psychiatric disorders.

Postsynaptic density (PSD) proteins have been implicated in the pathophysiology of neurodevelopmental and psychiatric disorders. Here, we present detailed clinical and genetic data for 20 patients with likely gene-disrupting mutations in TANC2-whose protein product interacts with multiple PSD proteins. Pediatric patients with disruptive mutations present with autism, intellectual disability, and delayed language and motor development. In addition to a variable degree of epilepsy and facial dysmorphism, we observe a pattern of more complex psychiatric dysfunction or behavioral problems in adult probands or carrier parents. Although this observation requires replication to establish statistical significance, it also suggests that mutations in this gene are associated with a variety of neuropsychiatric disorders consistent with its postsynaptic function. We find that TANC2 is expressed broadly in the human developing brain, especially in excitatory neurons and glial cells, but shows a more restricted pattern in Drosophila glial cells where its disruption affects behavioral outcomes.

Deleterious Variation in BRSK2 Associates with a Neurodevelopmental Disorder.

Developmental delay and intellectual disability (DD and ID) are heterogeneous phenotypes that arise in many rare monogenic disorders. Because of this rarity, developing cohorts with enough individuals to robustly identify disease-associated genes is challenging. Social-media platforms that facilitate data sharing among sequencing labs can help to address this challenge. Through one such tool, GeneMatcher, we identified nine DD- and/or ID-affected probands with a rare, heterozygous variant in the gene encoding the serine/threonine-protein kinase BRSK2. All probands have a speech delay, and most present with intellectual disability, motor delay, behavioral issues, and autism. Six of the nine variants are predicted to result in loss of function, and computational modeling predicts that the remaining three missense variants are damaging to BRSK2 structure and function. All nine variants are absent from large variant databases, and BRSK2 is, in general, relatively intolerant to protein-altering variation among humans. In all six probands for whom parents were available, the mutations were found to have arisen de novo. Five of these de novo variants were from cohorts with at least 400 sequenced probands; collectively, the cohorts span 3,429 probands, and the observed rate of de novo variation in these cohorts is significantly higher than the estimated background-mutation rate (p = 2.46 × 10-6). We also find that exome sequencing provides lower coverage and appears less sensitive to rare variation in BRSK2 than does genome sequencing; this fact most likely reduces BRSK2's visibility in many clinical and research sequencing efforts. Altogether, our results implicate damaging variation in BRSK2 as a source of neurodevelopmental disease.