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Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1, PSMC3, or PSMD12 proteasome genes. Proteasomes are large multi-subunit protein complexes that maintain cellular protein homeostasis by clearing ubiquitin-tagged damaged, misfolded, or unnecessary proteins. In this study, we have identified PSMD11 as an additional proteasome gene in which pathogenic variation is associated with an NDD-causing proteasomopathy. PSMD11 loss-of-function variants caused early-onset syndromic intellectual disability and neurodevelopmental delay with recurrent obesity in 10 unrelated children. Our findings demonstrate that the cognitive impairment observed in these individuals could be recapitulated in Drosophila melanogaster with depletion of the PMSD11 ortholog Rpn6, which compromised reversal learning. Our investigations in subject samples further revealed that PSMD11 loss of function resulted in impaired 26S proteasome assembly and the acquisition of a persistent type I interferon (IFN) gene signature, mediated by the integrated stress response (ISR) protein kinase R (PKR). In summary, these data identify PSMD11 as an additional member of the growing family of genes associated with neurodevelopmental proteasomopathies and provide insights into proteasomal biology in human health., Competing Interests: Declaration of interests A.C. and K.M. are employees of GeneDx, LLC. J.R.L. has stock in 23andMe and is a paid consultant for Genome International., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency., Competing Interests: Declaration of interests W.K.C. is a member of the Board of Directors of Prime Medicine and RallyBio. R.P. is an employee of GeneDx, LLC., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

De novo variants are a leading cause of neurodevelopmental disorders (NDDs), but because every monogenic NDD is different and usually extremely rare, it remains a major challenge to understand the complete phenotype and genotype spectrum of any morbid gene. According to OMIM, heterozygous variants in KDM6B cause "neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities." Here, by examining the molecular and clinical spectrum of 85 reported individuals with mostly de novo (likely) pathogenic KDM6B variants, we demonstrate that this description is inaccurate and potentially misleading. Cognitive deficits are seen consistently in all individuals, but the overall phenotype is highly variable. Notably, coarse facies and distal skeletal anomalies, as defined by OMIM, are rare in this expanded cohort while other features are unexpectedly common (e.g., hypotonia, psychosis, etc.). Using 3D protein structure analysis and an innovative dual Drosophila gain-of-function assay, we demonstrated a disruptive effect of 11 missense/in-frame indels located in or near the enzymatic JmJC or Zn-containing domain of KDM6B. Consistent with the role of KDM6B in human cognition, we demonstrated a role for the Drosophila KDM6B ortholog in memory and behavior. Taken together, we accurately define the broad clinical spectrum of the KDM6B-related NDD, introduce an innovative functional testing paradigm for the assessment of KDM6B variants, and demonstrate a conserved role for KDM6B in cognition and behavior. Our study demonstrates the critical importance of international collaboration, sharing of clinical data, and rigorous functional analysis of genetic variants to ensure correct disease diagnosis for rare disorders., Competing Interests: Declaration of interests S.W.S. is a scientific consultant of Population Bio and the King Abdullaziz University, and Athena Diagnostics has licensed intellectual property from his work held by the Hospital for Sick Children, Toronto., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Calcium (Ca 2+ ) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca 2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca 2+ pumps that participate in the regulation of intracellular free Ca 2+ . Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca 2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca 2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder., Competing Interests: Declaration of interests R.P. is an employee of GeneDx, Inc. All other authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies., (Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Although the role of typical Rho GTPases and other Rho-linked proteins in synaptic plasticity and cognitive function and dysfunction is widely acknowledged, the role of atypical Rho GTPases (such as RHOBTB2) in neurodevelopment has barely been characterized. We have now identified de novo missense variants clustering in the BTB-domain-encoding region of RHOBTB2 in ten individuals with a similar phenotype, including early-onset epilepsy, severe intellectual disability, postnatal microcephaly, and movement disorders. Three of the variants were recurrent. Upon transfection of HEK293 cells, we found that mutant RHOBTB2 was more abundant than the wild-type, most likely because of impaired degradation in the proteasome. Similarly, elevated amounts of the Drosophila ortholog RhoBTB in vivo were associated with seizure susceptibility and severe locomotor defects. Knockdown of RhoBTB in the Drosophila dendritic arborization neurons resulted in a decreased number of dendrites, thus suggesting a role of RhoBTB in dendritic development. We have established missense variants in the BTB-domain-encoding region of RHOBTB2 as causative for a developmental and epileptic encephalopathy and have elucidated the role of atypical Rho GTPase RhoBTB in Drosophila neurological function and possibly dendrite development., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

DHX30 is a member of the family of DExH-box helicases, which use ATP hydrolysis to unwind RNA secondary structures. Here we identified six different de novo missense mutations in DHX30 in twelve unrelated individuals affected by global developmental delay (GDD), intellectual disability (ID), severe speech impairment and gait abnormalities. While four mutations are recurrent, two are unique with one affecting the codon of one recurrent mutation. All amino acid changes are located within highly conserved helicase motifs and were found to either impair ATPase activity or RNA recognition in different in vitro assays. Moreover, protein variants exhibit an increased propensity to trigger stress granule (SG) formation resulting in global translation inhibition. Thus, our findings highlight the prominent role of translation control in development and function of the central nervous system and also provide molecular insight into how DHX30 dysfunction might cause a neurodevelopmental disorder., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The overall understanding of the molecular etiologies of intellectual disability (ID) and developmental delay (DD) is increasing as next-generation sequencing technologies identify genetic variants in individuals with such disorders. However, detailed analyses conclusively confirming these variants, as well as the underlying molecular mechanisms explaining the diseases, are often lacking. Here, we report on an ID syndrome caused by de novo heterozygous loss-of-function (LoF) mutations in SON. The syndrome is characterized by ID and/or DD, malformations of the cerebral cortex, epilepsy, vision problems, musculoskeletal abnormalities, and congenital malformations. Knockdown of son in zebrafish resulted in severe malformation of the spine, brain, and eyes. Importantly, analyses of RNA from affected individuals revealed that genes critical for neuronal migration and cortex organization (TUBG1, FLNA, PNKP, WDR62, PSMD3, and HDAC6) and metabolism (PCK2, PFKL, IDH2, ACY1, and ADA) are significantly downregulated because of the accumulation of mis-spliced transcripts resulting from erroneous SON-mediated RNA splicing. Our data highlight SON as a master regulator governing neurodevelopment and demonstrate the importance of SON-mediated RNA splicing in human development., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)