Publications by authors named "Tjitske Kleefstra"

162 Publications

SETD1A Mediated H3K4 Methylation and Its Role in Neurodevelopmental and Neuropsychiatric Disorders.

Front Mol Neurosci 2021 3;14:772000. Epub 2021 Nov 3.

Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, Nijmegen, Netherlands.

Posttranslational modification of histones and related gene regulation are shown to be affected in an increasing number of neurological disorders. SETD1A is a chromatin remodeler that influences gene expression through the modulation of mono- di- and trimethylation marks on Histone-H3-Lysine-4 (H3K4me1/2/3). H3K4 methylation is predominantly described to result in transcriptional activation, with its mono- di- and trimethylated forms differentially enriched at promoters or enhancers. Recently, dominant mostly variants in have clinically been linked to developmental delay, intellectual disability (DD/ID), and schizophrenia (SCZ). Affected individuals often display both developmental and neuropsychiatric abnormalities. The primary diagnoses are mainly dependent on the age at which the individual is assessed. Investigations in mouse models of SETD1A dysfunction have been able to recapitulate key behavioral features associated with ID and SCZ. Furthermore, functional investigations suggest disrupted synaptic and neuronal network function in these mouse models. In this review, we provide an overview of pre-clinical studies on the role of SETD1A in neuronal development. A better understanding of the pathobiology underlying these disorders may provide novel opportunities for therapeutic intervention. As such, we will discuss possible strategies to move forward in elucidating the genotype-phenotype correlation in associated disorders.
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http://dx.doi.org/10.3389/fnmol.2021.772000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8595121PMC
November 2021

Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro.

Stem Cell Reports 2021 Sep 29;16(9):2182-2196. Epub 2021 Jul 29.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behavior, 6500 HB Nijmegen, the Netherlands; Department of Clinical Genomics, Mayo Clinic, Rochester, MN 55905, USA. Electronic address:

Micro-electrode arrays (MEAs) are increasingly used to characterize neuronal network activity of human induced pluripotent stem cell (hiPSC)-derived neurons. Despite their gain in popularity, MEA recordings from hiPSC-derived neuronal networks are not always used to their full potential in respect to experimental design, execution, and data analysis. Therefore, we benchmarked the robustness of MEA-derived neuronal activity patterns from ten healthy individual control lines, and uncover comparable network phenotypes. To achieve standardization, we provide recommendations on experimental design and analysis. With such standardization, MEAs can be used as a reliable platform to distinguish (disease-specific) network phenotypes. In conclusion, we show that MEAs are a powerful and robust tool to uncover functional neuronal network phenotypes from hiPSC-derived neuronal networks, and provide an important resource to advance the hiPSC field toward the use of MEAs for disease phenotyping and drug discovery.
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http://dx.doi.org/10.1016/j.stemcr.2021.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452490PMC
September 2021

Speech-language profiles in the context of cognitive and adaptive functioning in SATB2-associated syndrome.

Genes Brain Behav 2021 09;20(7):e12761

Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

SATB2-associated syndrome (SAS) is a neurodevelopmental disorder caused by heterozygous pathogenic variants in the SATB2 gene, and is typically characterized by intellectual disability and severely impaired communication skills. The goal of this study was to contribute to the understanding of speech and language impairments in SAS, in the context of general developmental skills and cognitive and adaptive functioning. We performed detailed oral motor, speech and language profiling in combination with neuropsychological assessments in 23 individuals with a molecularly confirmed SAS diagnosis: 11 primarily verbal individuals and 12 primarily nonverbal individuals, independent of their ages. All individuals had severe receptive language delays. For all verbal individuals, we were able to define underlying speech conditions. While childhood apraxia of speech was most prevalent, oral motor problems appeared frequent as well and were more present in the nonverbal group than in the verbal group. For seven individuals, age-appropriate Wechsler indices could be derived, showing that the level of intellectual functioning of these individuals varied from moderate-mild ID to mild ID-borderline intellectual functioning. Assessments of adaptive functioning with the Vineland Screener showed relatively high scores on the domain "daily functioning" and relatively low scores on the domain "communication" in most individuals. Altogether, this study provides a detailed delineation of oral motor, speech and language skills and neuropsychological functioning in individuals with SAS, and can provide families and caregivers with information to guide diagnosis, management and treatment approaches.
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http://dx.doi.org/10.1111/gbb.12761DOI Listing
September 2021

The CHD8/CHD7/Kismet family links blood-brain barrier glia and serotonin to ASD-associated sleep defects.

Sci Adv 2021 Jun 4;7(23). Epub 2021 Jun 4.

Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud university medical center, 6525 GA, Nijmegen, Netherlands.

Sleep disturbances in autism and neurodevelopmental disorders are common and adversely affect patient's quality of life, yet the underlying mechanisms are understudied. We found that individuals with mutations in , among the highest-confidence autism risk genes, or suffer from disturbed sleep maintenance. These defects are recapitulated in mutants affecting , the sole ortholog. We show that Kismet is required in glia for early developmental and adult sleep architecture. This role localizes to subperineurial glia constituting the blood-brain barrier. We demonstrate that Kismet-related sleep disturbances are caused by high serotonin during development, paralleling a well-established but genetically unsolved autism endophenotype. Despite their developmental origin, Kismet's sleep architecture defects can be reversed in adulthood by a behavioral regime resembling human sleep restriction therapy. Our findings provide fundamental insights into glial regulation of sleep and propose a causal mechanistic link between the CHD8/CHD7/Kismet family, developmental hyperserotonemia, and autism-associated sleep disturbances.
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http://dx.doi.org/10.1126/sciadv.abe2626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177706PMC
June 2021

A MT-TL1 variant identified by whole exome sequencing in an individual with intellectual disability, epilepsy, and spastic tetraparesis.

Eur J Hum Genet 2021 Sep 1;29(9):1359-1368. Epub 2021 Jun 1.

Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands.

The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line.
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http://dx.doi.org/10.1038/s41431-021-00900-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8440635PMC
September 2021

De novo variants in TCF7L2 are associated with a syndromic neurodevelopmental disorder.

Am J Med Genet A 2021 08 18;185(8):2384-2390. Epub 2021 May 18.

Division of Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

TCF7L2 encodes transcription factor 7-like 2 (OMIM 602228), a key mediator of the evolutionary conserved canonical Wnt signaling pathway. Although several large-scale sequencing studies have implicated TCF7L2 in intellectual disability and autism, both the genetic mechanism and clinical phenotype have remained incompletely characterized. We present here a comprehensive genetic and phenotypic description of 11 individuals who have been identified to carry de novo variants in TCF7L2, both truncating and missense. Missense variation is clustered in or near a high mobility group box domain, involving this region in these variants' pathogenicity. All affected individuals present with developmental delays in childhood, but most ultimately achieved normal intelligence or had only mild intellectual disability. Myopia was present in approximately half of the individuals, and some individuals also possessed dysmorphic craniofacial features, orthopedic abnormalities, or neuropsychiatric comorbidities including autism and attention-deficit/hyperactivity disorder (ADHD). We thus present an initial clinical and genotypic spectrum associated with variation in TCF7L2, which will be important in informing both medical management and future research.
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http://dx.doi.org/10.1002/ajmg.a.62254DOI Listing
August 2021

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphism.

Am J Hum Genet 2021 06 27;108(6):1138-1150. Epub 2021 Apr 27.

Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA.

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder.
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http://dx.doi.org/10.1016/j.ajhg.2021.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206162PMC
June 2021

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature.

Am J Hum Genet 2021 06 27;108(6):1053-1068. Epub 2021 Apr 27.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany.

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.
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http://dx.doi.org/10.1016/j.ajhg.2021.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206150PMC
June 2021

Clinical delineation of SETBP1 haploinsufficiency disorder.

Eur J Hum Genet 2021 Aug 19;29(8):1198-1205. Epub 2021 Apr 19.

Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.

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.
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http://dx.doi.org/10.1038/s41431-021-00888-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385049PMC
August 2021

TAOK1 is associated with neurodevelopmental disorder and essential for neuronal maturation and cortical development.

Hum Mutat 2021 Apr 1;42(4):445-459. Epub 2021 Mar 1.

Department of Pediatrics, SIU School of Medicine, Springfield, Illinois, USA.

Thousand and one amino-acid kinase 1 (TAOK1) is a MAP3K protein kinase, regulating different mitogen-activated protein kinase pathways, thereby modulating a multitude of processes in the cell. Given the recent finding of TAOK1 involvement in neurodevelopmental disorders (NDDs), we investigated the role of TAOK1 in neuronal function and collected a cohort of 23 individuals with mostly de novo variants in TAOK1 to further define the associated NDD. Here, we provide evidence for an important role for TAOK1 in neuronal function, showing that altered TAOK1 expression levels in the embryonic mouse brain affect neural migration in vivo, as well as neuronal maturation in vitro. The molecular spectrum of the identified TAOK1 variants comprises largely truncating and nonsense variants, but also missense variants, for which we provide evidence that they can have a loss of function or dominant-negative effect on TAOK1, expanding the potential underlying causative mechanisms resulting in NDD. Taken together, our data indicate that TAOK1 activity needs to be properly controlled for normal neuronal function and that TAOK1 dysregulation leads to a neurodevelopmental disorder mainly comprising similar facial features, developmental delay/intellectual disability and/or variable learning or behavioral problems, muscular hypotonia, infant feeding difficulties, and growth problems.
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http://dx.doi.org/10.1002/humu.24176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248425PMC
April 2021

Mutation-specific pathophysiological mechanisms define different neurodevelopmental disorders associated with SATB1 dysfunction.

Am J Hum Genet 2021 02 28;108(2):346-356. Epub 2021 Jan 28.

Department of Rehabilitation and Development, Randall Children's Hospital at Legacy Emanuel Medical Center, Portland, OR 97227, USA.

Whereas large-scale statistical analyses can robustly identify disease-gene relationships, they do not accurately capture genotype-phenotype correlations or disease mechanisms. We use multiple lines of independent evidence to show that different variant types in a single gene, SATB1, cause clinically overlapping but distinct neurodevelopmental disorders. Clinical evaluation of 42 individuals carrying SATB1 variants identified overt genotype-phenotype relationships, associated with different pathophysiological mechanisms, established by functional assays. Missense variants in the CUT1 and CUT2 DNA-binding domains result in stronger chromatin binding, increased transcriptional repression, and a severe phenotype. In contrast, variants predicted to result in haploinsufficiency are associated with a milder clinical presentation. A similarly mild phenotype is observed for individuals with premature protein truncating variants that escape nonsense-mediated decay, which are transcriptionally active but mislocalized in the cell. Our results suggest that in-depth mutation-specific genotype-phenotype studies are essential to capture full disease complexity and to explain phenotypic variability.
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http://dx.doi.org/10.1016/j.ajhg.2021.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895900PMC
February 2021

Comprehensive study of 28 individuals with SIN3A-related disorder underscoring the associated mild cognitive and distinctive facial phenotype.

Eur J Hum Genet 2021 04 12;29(4):625-636. Epub 2021 Jan 12.

Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, the Netherlands.

Witteveen-Kolk syndrome (OMIM 613406) is a recently defined neurodevelopmental syndrome caused by heterozygous loss-of-function variants in SIN3A. We define the clinical and neurodevelopmental phenotypes related to SIN3A-haploinsufficiency in 28 unreported patients. Patients with SIN3A variants adversely affecting protein function have mild intellectual disability, growth and feeding difficulties. Involvement of a multidisciplinary team including a geneticist, paediatrician and neurologist should be considered in managing these patients. Patients described here were identified through a combination of clinical evaluation and gene matching strategies (GeneMatcher and Decipher). All patients consented to participate in this study. Mean age of this cohort was 8.2 years (17 males, 11 females). Out of 16 patients ≥ 8 years old assessed, eight (50%) had mild intellectual disability (ID), four had moderate ID (22%), and one had severe ID (6%). Four (25%) did not have any cognitive impairment. Other neurological symptoms such as seizures (4/28) and hypotonia (12/28) were common. Behaviour problems were reported in a minority. In patients ≥2 years, three were diagnosed with Autism Spectrum Disorder (ASD) and four with Attention Deficit Hyperactivity Disorder (ADHD). We report 27 novel variants and one previously reported variant. 24 were truncating variants; three were missense variants and one large in-frame gain including exons 10-12.
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http://dx.doi.org/10.1038/s41431-020-00769-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115148PMC
April 2021

Haploinsufficiency of the HIRA gene located in the 22q11 deletion syndrome region is associated with abnormal neurodevelopment and impaired dendritic outgrowth.

Hum Genet 2021 Jun 8;140(6):885-896. Epub 2021 Jan 8.

Service de Génétique, Centre Hospitalier Régional Universitaire, Tours, France.

The 22q11.2 deletion syndrome (22q11DS) is associated with a wide spectrum of cognitive and psychiatric symptoms. Despite the considerable work performed over the past 20 years, the genetic etiology of the neurodevelopmental phenotype remains speculative. Here, we report de novo heterozygous truncating variants in the HIRA (Histone cell cycle regulation defective, S. Cerevisiae, homolog of, A) gene associated with a neurodevelopmental disorder in two unrelated patients. HIRA is located within the commonly deleted region of the 22q11DS and encodes a histone chaperone that regulates neural progenitor proliferation and neurogenesis, and that belongs to the WD40 Repeat (WDR) protein family involved in brain development and neuronal connectivity. To address the specific impact of HIRA haploinsufficiency in the neurodevelopmental phenotype of 22q11DS, we combined Hira knock-down strategies in developing mouse primary hippocampal neurons, and the direct study of brains from heterozygous Hira mice. Our in vitro analyses revealed that Hira gene is mostly expressed during neuritogenesis and early dendritogenesis stages in mouse total brain and in developing primary hippocampal neurons. Moreover, shRNA knock-down experiments showed that a twofold decrease of endogenous Hira expression level resulted in an impaired dendritic growth and branching in primary developing hippocampal neuronal cultures. In parallel, in vivo analyses demonstrated that Hira mice displayed subtle neuroanatomical defects including a reduced size of the hippocampus, the fornix and the corpus callosum. Our results suggest that HIRA haploinsufficiency would likely contribute to the complex pathophysiology of the neurodevelopmental phenotype of 22q11DS by impairing key processes in neurogenesis and by causing neuroanatomical defects during cerebral development.
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http://dx.doi.org/10.1007/s00439-020-02252-1DOI Listing
June 2021

Missense variant contribution to USP9X-female syndrome.

NPJ Genom Med 2020 Dec 9;5(1):53. Epub 2020 Dec 9.

Griffith Institute for Drug Discovery, Griffith University, Brisbane, QLD, Australia.

USP9X is an X-chromosome gene that escapes X-inactivation. Loss or compromised function of USP9X leads to neurodevelopmental disorders in males and females. While males are impacted primarily by hemizygous partial loss-of-function missense variants, in females de novo heterozygous complete loss-of-function mutations predominate, and give rise to the clinically recognisable USP9X-female syndrome. Here we provide evidence of the contribution of USP9X missense and small in-frame deletion variants in USP9X-female syndrome also. We scrutinise the pathogenicity of eleven such variants, ten of which were novel. Combined application of variant prediction algorithms, protein structure modelling, and assessment under clinically relevant guidelines universally support their pathogenicity. The core phenotype of this cohort overlapped with previous descriptions of USP9X-female syndrome, but exposed heightened variability. Aggregate phenotypic information of 35 currently known females with predicted pathogenic variation in USP9X reaffirms the clinically recognisable USP9X-female syndrome, and highlights major differences when compared to USP9X-male associated neurodevelopmental disorders.
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http://dx.doi.org/10.1038/s41525-020-00162-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725775PMC
December 2020

Germline AGO2 mutations impair RNA interference and human neurological development.

Nat Commun 2020 11 16;11(1):5797. Epub 2020 Nov 16.

Department of Genetics, Children's Hospital of Eastern Ontario, Ottawa, ON, Canada.

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.
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http://dx.doi.org/10.1038/s41467-020-19572-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670403PMC
November 2020

Heterozygous variants that disturb the transcriptional repressor activity of FOXP4 cause a developmental disorder with speech/language delays and multiple congenital abnormalities.

Genet Med 2021 03 28;23(3):534-542. Epub 2020 Oct 28.

Language & Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen, The Netherlands.

Purpose: Heterozygous pathogenic variants in various FOXP genes cause specific developmental disorders. The phenotype associated with heterozygous variants in FOXP4 has not been previously described.

Methods: We assembled a cohort of eight individuals with heterozygous and mostly de novo variants in FOXP4: seven individuals with six different missense variants and one individual with a frameshift variant. We collected clinical data to delineate the phenotypic spectrum, and used in silico analyses and functional cell-based assays to assess pathogenicity of the variants.

Results: We collected clinical data for six individuals: five individuals with a missense variant in the forkhead box DNA-binding domain of FOXP4, and one individual with a truncating variant. Overlapping features included speech and language delays, growth abnormalities, congenital diaphragmatic hernia, cervical spine abnormalities, and ptosis. Luciferase assays showed loss-of-function effects for all these variants, and aberrant subcellular localization patterns were seen in a subset. The remaining two missense variants were located outside the functional domains of FOXP4, and showed transcriptional repressor capacities and localization patterns similar to the wild-type protein.

Conclusion: Collectively, our findings show that heterozygous loss-of-function variants in FOXP4 are associated with an autosomal dominant neurodevelopmental disorder with speech/language delays, growth defects, and variable congenital abnormalities.
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http://dx.doi.org/10.1038/s41436-020-01016-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935712PMC
March 2021

Behavior and cognitive functioning in Witteveen-Kolk syndrome.

Am J Med Genet A 2020 10 11;182(10):2384-2390. Epub 2020 Aug 11.

Center of Excellence for Neuropsychiatry, Vincent van Gogh Institute for Psychiatry, Venray, The Netherlands.

Witteveen-Kolk syndrome (WITKOS) is a rare neurodevelopmental disorder characterized by developmental delay/intellectual disability, facial dysmorphisms, and short stature. The syndrome is caused by loss of function of switch-insensitive 3 transcription regulator family member A (SIN3A). Regarding behavioral functioning, Autism Spectrum Disorders (ASD), obsessive-compulsive behaviors, as well as Attention-Deficit/Hyperactivity Disorder symptoms (ADHD) have been suggested. The present study explores various aspects of neurocognitive functioning in five individuals (age range 10-23) with WITKOS. Medical records and results of extensive neuropsychological assessment are used to describe developmental trajectories and neurocognitive profiles. Systematic analysis of medical records displays developmental difficulties described as ASD or ADHD in childhood, sleep problems and internalizing problems during adolescence. Results of cognitive assessments indicate profoundly disabled (n = 1), mildly disabled (n = 2), borderline (n = 1), and average (n = 1) levels of intelligence. Furthermore, results indicate weaknesses in speed of information processing/sustained attention in all participants, and difficulties in planning and maintaining overview in three participants. Furthermore, parent reports of behavioral functioning primarily suggest problems in social functioning. Implications of both cognitive problems and social-emotional vulnerabilities for counseling are discussed and supplemented with suggestions for interventions.
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http://dx.doi.org/10.1002/ajmg.a.61775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540409PMC
October 2020

De Novo Variants in CNOT1, a Central Component of the CCR4-NOT Complex Involved in Gene Expression and RNA and Protein Stability, Cause Neurodevelopmental Delay.

Am J Hum Genet 2020 07 17;107(1):164-172. Epub 2020 Jun 17.

Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.
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http://dx.doi.org/10.1016/j.ajhg.2020.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332645PMC
July 2020

Characterization of SETD1A haploinsufficiency in humans and Drosophila defines a novel neurodevelopmental syndrome.

Mol Psychiatry 2021 06 28;26(6):2013-2024. Epub 2020 Apr 28.

Center for Pediatric Genomic Medicine, Children's Mercy Hospital, University of Missouri-Kansas City School of Medicine, Kansas City, MO, USA.

Defects in histone methyltransferases (HMTs) are major contributing factors in neurodevelopmental disorders (NDDs). Heterozygous variants of SETD1A involved in histone H3 lysine 4 (H3K4) methylation were previously identified in individuals with schizophrenia. Here, we define the clinical features of the Mendelian syndrome associated with haploinsufficiency of SETD1A by investigating 15 predominantly pediatric individuals who all have de novo SETD1A variants. These individuals present with a core set of symptoms comprising global developmental delay and/or intellectual disability, subtle facial dysmorphisms, behavioral and psychiatric problems. We examined cellular phenotypes in three patient-derived lymphoblastoid cell lines with three variants: p.Gly535Alafs*12, c.4582-2_4582delAG, and p.Tyr1499Asp. These patient cell lines displayed DNA damage repair defects that were comparable to previously observed RNAi-mediated depletion of SETD1A. This suggested that these variants, including the p.Tyr1499Asp in the catalytic SET domain, behave as loss-of-function (LoF) alleles. Previous studies demonstrated a role for SETD1A in cell cycle control and differentiation. However, individuals with SETD1A variants do not show major structural brain defects or severe microcephaly, suggesting that defective proliferation and differentiation of neural progenitors is unlikely the single underlying cause of the disorder. We show here that the Drosophila melanogaster SETD1A orthologue is required in postmitotic neurons of the fly brain for normal memory, suggesting a role in post development neuronal function. Together, this study defines a neurodevelopmental disorder caused by dominant de novo LoF variants in SETD1A and further supports a role for H3K4 methyltransferases in the regulation of neuronal processes underlying normal cognitive functioning.
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http://dx.doi.org/10.1038/s41380-020-0725-5DOI Listing
June 2021

Damaging de novo missense variants in EEF1A2 lead to a developmental and degenerative epileptic-dyskinetic encephalopathy.

Hum Mutat 2020 07 6;41(7):1263-1279. Epub 2020 Apr 6.

Department of Genetics, Utrecht University, Utrecht, The Netherlands.

Heterozygous de novo variants in the eukaryotic elongation factor EEF1A2 have previously been described in association with intellectual disability and epilepsy but never functionally validated. Here we report 14 new individuals with heterozygous EEF1A2 variants. We functionally validate multiple variants as protein-damaging using heterologous expression and complementation analysis. Our findings allow us to confirm multiple variants as pathogenic and broaden the phenotypic spectrum to include dystonia/choreoathetosis, and in some cases a degenerative course with cerebral and cerebellar atrophy. Pathogenic variants appear to act via a haploinsufficiency mechanism, disrupting both the protein synthesis and integrated stress response functions of EEF1A2. Our studies provide evidence that EEF1A2 is highly intolerant to variation and that de novo pathogenic variants lead to an epileptic-dyskinetic encephalopathy with both neurodevelopmental and neurodegenerative features. Developmental features may be driven by impaired synaptic protein synthesis during early brain development while progressive symptoms may be linked to an impaired ability to handle cytotoxic stressors.
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http://dx.doi.org/10.1002/humu.24015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292794PMC
July 2020

Evaluation of DNA Methylation Episignatures for Diagnosis and Phenotype Correlations in 42 Mendelian Neurodevelopmental Disorders.

Am J Hum Genet 2020 03 27;106(3):356-370. Epub 2020 Feb 27.

Université de Paris, Epigénétique et Destin Cellulaire, CNRS, 75013 Paris, France.

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called "episignatures"). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders.
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http://dx.doi.org/10.1016/j.ajhg.2020.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058829PMC
March 2020

Distinct Pathogenic Genes Causing Intellectual Disability and Autism Exhibit a Common Neuronal Network Hyperactivity Phenotype.

Cell Rep 2020 01;30(1):173-186.e6

Department of Cognitive Neuroscience, Radboudumc, Donders Institute for Brain, Cognition and Behaviour, 6525 HR Nijmegen, the Netherlands; Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500 HB Nijmegen, the Netherlands. Electronic address:

Pathogenic mutations in either one of the epigenetic modifiers EHMT1, MBD5, MLL3, or SMARCB1 have been identified to be causative for Kleefstra syndrome spectrum (KSS), a neurodevelopmental disorder with clinical features of both intellectual disability (ID) and autism spectrum disorder (ASD). To understand how these variants lead to the phenotypic convergence in KSS, we employ a loss-of-function approach to assess neuronal network development at the molecular, single-cell, and network activity level. KSS-gene-deficient neuronal networks all develop into hyperactive networks with altered network organization and excitatory-inhibitory balance. Interestingly, even though transcriptional data reveal distinct regulatory mechanisms, KSS target genes share similar functions in regulating neuronal excitability and synaptic function, several of which are associated with ID and ASD. Our results show that KSS genes mainly converge at the level of neuronal network communication, providing insights into the pathophysiology of KSS and phenotypically congruent disorders.
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http://dx.doi.org/10.1016/j.celrep.2019.12.002DOI Listing
January 2020

Neuronal network dysfunction in a model for Kleefstra syndrome mediated by enhanced NMDAR signaling.

Nat Commun 2019 10 30;10(1):4928. Epub 2019 Oct 30.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500, HB Nijmegen, Netherlands.

Kleefstra syndrome (KS) is a neurodevelopmental disorder caused by mutations in the histone methyltransferase EHMT1. To study the impact of decreased EHMT1 function in human cells, we generated excitatory cortical neurons from induced pluripotent stem (iPS) cells derived from KS patients. Neuronal networks of patient-derived cells exhibit network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes are mediated by upregulation of NMDA receptor (NMDAR) subunit 1 correlating with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. In mice EHMT1 deficiency leads to similar neuronal network impairments with increased NMDAR function. Finally, we rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Summarized, we demonstrate a direct link between EHMT1 deficiency and NMDAR hyperfunction in human neurons, providing a potential basis for more targeted therapeutic approaches for KS.
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http://dx.doi.org/10.1038/s41467-019-12947-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821803PMC
October 2019

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

Nat Commun 2019 10 15;10(1):4679. Epub 2019 Oct 15.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.

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.
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http://dx.doi.org/10.1038/s41467-019-12435-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794285PMC
October 2019

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling.

Biol Psychiatry 2020 01 29;87(2):100-112. Epub 2019 Jun 29.

Institute of Human Genetics, Heidelberg University, Heidelberg, Germany.

Background: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative.

Methods: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology.

Results: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory.

Conclusions: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function.
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http://dx.doi.org/10.1016/j.biopsych.2019.05.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925349PMC
January 2020

Enabling Global Clinical Collaborations on Identifiable Patient Data: The Minerva Initiative.

Front Genet 2019 29;10:611. Epub 2019 Jul 29.

Department of Medical Genetics, University of Antwerp, Antwerp, Belgium.

The clinical utility of computational phenotyping for both genetic and rare diseases is increasingly appreciated; however, its true potential is yet to be fully realized. Alongside the growing clinical and research availability of sequencing technologies, precise deep and scalable phenotyping is required to serve unmet need in genetic and rare diseases. To improve the lives of individuals affected with rare diseases through deep phenotyping, global big data interrogation is necessary to aid our understanding of disease biology, assist diagnosis, and develop targeted treatment strategies. This includes the application of cutting-edge machine learning methods to image data. As with most digital tools employed in health care, there are ethical and data governance challenges associated with using identifiable personal image data. There are also risks with failing to deliver on the patient benefits of these new technologies, the biggest of which is posed by data siloing. The Minerva Initiative has been designed to enable the public good of deep phenotyping while mitigating these ethical risks. Its open structure, enabling collaboration and data sharing between individuals, clinicians, researchers and private enterprise, is key for delivering precision public health.
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http://dx.doi.org/10.3389/fgene.2019.00611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681681PMC
July 2019

Mutations in PIGU Impair the Function of the GPI Transamidase Complex, Causing Severe Intellectual Disability, Epilepsy, and Brain Anomalies.

Am J Hum Genet 2019 08 25;105(2):395-402. Epub 2019 Jul 25.

Institute for Genomic Statistics and Bioinformatics, University Hospital Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, 53127 Bonn, Germany.

The glycosylphosphatidylinositol (GPI) anchor links over 150 proteins to the cell surface and is present on every cell type. Many of these proteins play crucial roles in neuronal development and function. Mutations in 18 of the 29 genes implicated in the biosynthesis of the GPI anchor have been identified as the cause of GPI biosynthesis deficiencies (GPIBDs) in humans. GPIBDs are associated with intellectual disability and seizures as their cardinal features. An essential component of the GPI transamidase complex is PIGU, along with PIGK, PIGS, PIGT, and GPAA1, all of which link GPI-anchored proteins (GPI-APs) onto the GPI anchor in the endoplasmic reticulum (ER). Here, we report two homozygous missense mutations (c.209T>A [p.Ile70Lys] and c.1149C>A [p.Asn383Lys]) in five individuals from three unrelated families. All individuals presented with global developmental delay, severe-to-profound intellectual disability, muscular hypotonia, seizures, brain anomalies, scoliosis, and mild facial dysmorphism. Using multicolor flow cytometry, we determined a characteristic profile for GPI transamidase deficiency. On granulocytes this profile consisted of reduced cell-surface expression of fluorescein-labeled proaerolysin (FLAER), CD16, and CD24, but not of CD55 and CD59; additionally, B cells showed an increased expression of free GPI anchors determined by T5 antibody. Moreover, computer-assisted facial analysis of different GPIBDs revealed a characteristic facial gestalt shared among individuals with mutations in PIGU and GPAA1. Our findings improve our understanding of the role of the GPI transamidase complex in the development of nervous and skeletal systems and expand the clinical spectrum of disorders belonging to the group of inherited GPI-anchor deficiencies.
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http://dx.doi.org/10.1016/j.ajhg.2019.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698879PMC
August 2019
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