Ash1 l loss-of-function results in structural birth defects and altered cortical development.

The histone methyltransferase ASH1L plays a crucial role in regulating gene expression across various organ systems during development, yet its role in brain development remains largely unexplored. Over 130 individuals with autism harbour heterozygous loss-of-function ASH1L variants, and population studies confirm it as a high-risk autism gene. Previous studies on Ash1 l deficient mice have reported autistic-like behaviours and provided insights into the underlying neuropathophysiology. In this study, we used mice with a cre-inducible deletion of Ash1 l exon 4, which results in a frame shift and premature stop codon (p.V1693Afs*2). Our investigation evaluated the impact of Ash1 l loss-of-function on survival and craniofacial skeletal development. Using a tamoxifen-inducible cre strain, we targeted Ash1 l knockout early in cortical development (Emx1-Cre-ERT2; e10.5). Immunohistochemistry was utilized to assess cortical lamination, while EdU incorporation aided in birthdating cortical neurons. Additionally, single-cell RNA sequencing was employed to compare cortical cell populations and identify genes with differential expression. At e18.5, the proportion of homozygous Ash1 l germline knockout embryos appeared normal; however, no live Ash1 l null pups were present at birth (e18.5: n = 77, P = 0.90; p0: n = 41, P = 0.00095). Notably, Ash1l-/- exhibited shortened nasal bones (n = 31, P = 0.017). In the cortical-specific knockout model, SATB2 neurons showed increased numbers (n = 6/genotype, P = 0.0001) and were distributed through the cortical plate. Birthdating revealed generation of ectopically placed deep layer neurons that express SATB2 (e13.5 injection: n = 4/genotype, P = 0.0126). Single cell RNA sequencing revealed significant differences in gene expression between control and mutant upper layer neurons, leading to distinct clustering. Pseudotime analysis indicated that the mutant cluster followed an altered cell differentiation trajectory. This study underscores the essential role of Ash1 l in postnatal survival and normal craniofacial development. In the cortex, ASH1L exerts broad effects on gene expression and is indispensable for determining the fate of upper layer cortical neurons. These findings provide valuable insights into the potential mechanisms of ASH1L neuropathology, shedding light on its significance in neurodevelopmental disorders like autism.

SRD5A3 is required for converting polyprenol to dolichol and is mutated in a congenital glycosylation disorder.

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.

H2A monoubiquitination: insights from human genetics and animal models.

Metazoan development arises from spatiotemporal control of gene expression, which depends on epigenetic regulators like the polycomb group proteins (PcG) that govern the chromatin landscape. PcG proteins facilitate the addition and removal of histone 2A monoubiquitination at lysine 119 (H2AK119ub1), which regulates gene expression, cell fate decisions, cell cycle progression, and DNA damage repair. Regulation of these processes by PcG proteins is necessary for proper development, as pathogenic variants in these genes are increasingly recognized to underly developmental disorders. Overlapping features of developmental syndromes associated with pathogenic variants in specific PcG genes suggest disruption of central developmental mechanisms; however, unique clinical features observed in each syndrome suggest additional non-redundant functions for each PcG gene. In this review, we describe the clinical manifestations of pathogenic PcG gene variants, review what is known about the molecular functions of these gene products during development, and interpret the clinical data to summarize the current evidence toward an understanding of the genetic and molecular mechanism.

A data set of variants derived from 1455 clinical and research exomes is efficient in variant prioritization for early-onset monogenic disorders in Indians.

Given the genomic uniqueness, a local data set is most desired for Indians, who are underrepresented in existing public databases. We hypothesize patients with rare monogenic disorders and their family members can provide a reliable source of common variants in the population. Exome sequencing (ES) data from families with rare Mendelian disorders was aggregated from five centers in India. The dataset was refined by excluding related individuals and removing the disease-causing variants (refined cohort). The efficiency of these data sets was assessed in a new set of 50 exomes against gnomAD and GenomeAsia. Our original cohort comprised 1455 individuals from 1203 families. The refined cohort had 836 unrelated individuals that retained 1,251,064 variants with 181,125 population-specific and 489,618 common variants. The allele frequencies from our cohort helped to define 97,609 rare variants in gnomAD and 44,520 rare variants in GenomeAsia as common variants in our population. Our variant dataset provided an additional 1.7% and 0.1% efficiency for prioritizing heterozygous and homozygous variants respectively for rare monogenic disorders. We observed additional 19 genes/human knockouts. We list carrier frequency for 142 recessive disorders. This is a large and useful resource of exonic variants for Indians. Despite limitations, datasets from patients are efficient tools for variant prioritization in a resource-limited setting.

Histone H2A Monoubiquitination in Neurodevelopmental Disorders.

Covalent histone modifications play an essential role in gene regulation and cellular specification required for multicellular organism development. Monoubiquitination of histone H2A (H2AUb1) is a reversible transcriptionally repressive mark. Exchange of histone H2A monoubiquitination and deubiquitination reflects the succession of transcriptional profiles during development required to produce cellular diversity from pluripotent cells. Germ-line pathogenic variants in components of the H2AUb1 regulatory axis are being identified as the genetic basis of congenital neurodevelopmental disorders. Here, we review the human genetics findings coalescing on molecular mechanisms that alter the genome-wide distribution of this histone modification required for development.

Mutations in Citron Kinase Cause Recessive Microlissencephaly with Multinucleated Neurons.

Primary microcephaly is a neurodevelopmental disorder that is caused by a reduction in brain size as a result of defects in the proliferation of neural progenitor cells during development. Mutations in genes encoding proteins that localize to the mitotic spindle and centrosomes have been implicated in the pathogenicity of primary microcephaly. In contrast, the contractile ring and midbody required for cytokinesis, the final stage of mitosis, have not previously been implicated by human genetics in the molecular mechanisms of this phenotype. Citron kinase (CIT) is a multi-domain protein that localizes to the cleavage furrow and midbody of mitotic cells, where it is required for the completion of cytokinesis. Rodent models of Cit deficiency highlighted the role of this gene in neurogenesis and microcephaly over a decade ago. Here, we identify recessively inherited pathogenic variants in CIT as the genetic basis of severe microcephaly and neonatal death. We present postmortem data showing that CIT is critical to building a normally sized human brain. Consistent with cytokinesis defects attributed to CIT, multinucleated neurons were observed throughout the cerebral cortex and cerebellum of an affected proband, expanding our understanding of mechanisms attributed to primary microcephaly.

Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly.

Cell division terminates with cytokinesis and cellular separation. Autosomal-recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a reduction in brain and head size at birth in addition to non-progressive intellectual disability. MCPH is genetically heterogeneous, and 16 loci are known to be associated with loss-of-function mutations predominantly affecting centrosomal-associated proteins, but the multiple roles of centrosomes in cellular function has left questions about etiology. Here, we identified three families affected by homozygous missense mutations in CIT, encoding citron rho-interacting kinase (CIT), which has established roles in cytokinesis. All mutations caused substitution of conserved amino acid residues in the kinase domain and impaired kinase activity. Neural progenitors that were differentiated from induced pluripotent stem cells (iPSCs) derived from individuals with these mutations exhibited abnormal cytokinesis with delayed mitosis, multipolar spindles, and increased apoptosis, rescued by CRISPR/Cas9 genome editing. Our results highlight the importance of cytokinesis in the pathology of primary microcephaly.

Genotype-phenotype correlations in individuals with pathogenic RERE variants.

Heterozygous variants in the arginine-glutamic acid dipeptide repeats gene (RERE) have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH). Here, we report nine individuals with NEDBEH who carry partial deletions or deleterious sequence variants in RERE. These variants were found to be de novo in all cases in which parental samples were available. An analysis of data from individuals with NEDBEH suggests that point mutations affecting the Atrophin-1 domain of RERE are associated with an increased risk of structural eye defects, congenital heart defects, renal anomalies, and sensorineural hearing loss when compared with loss-of-function variants that are likely to lead to haploinsufficiency. A high percentage of RERE pathogenic variants affect a histidine-rich region in the Atrophin-1 domain. We have also identified a recurrent two-amino-acid duplication in this region that is associated with the development of a CHARGE syndrome-like phenotype. We conclude that mutations affecting RERE result in a spectrum of clinical phenotypes. Genotype-phenotype correlations exist and can be used to guide medical decision making. Consideration should also be given to screening for RERE variants in individuals who fulfill diagnostic criteria for CHARGE syndrome but do not carry pathogenic variants in CHD7.

De novo dominant ASXL3 mutations alter H2A deubiquitination and transcription in Bainbridge-Ropers syndrome.

De novo truncating mutations in Additional sex combs-like 3 (ASXL3) have been identified in individuals with Bainbridge-Ropers syndrome (BRS), characterized by failure to thrive, global developmental delay, feeding problems, hypotonia, dysmorphic features, profound speech delays and intellectual disability. We identified three novel de novo heterozygous truncating variants distributed across ASXL3, outside the original cluster of ASXL3 mutations previously described for BRS. Primary skin fibroblasts established from a BRS patient were used to investigate the functional impact of pathogenic variants. ASXL3 mRNA transcripts from the mutated allele are prone to nonsense-mediated decay, and expression of ASXL3 is reduced. We found that ASXL3 interacts with BAP1, a hydrolase that removes mono-ubiquitin from histone H2A lysine 119 (H2AK119Ub1) as a component of the Polycomb repressive deubiquitination (PR-DUB) complex. A significant increase in H2AK119Ub1 was observed in ASXL3 patient fibroblasts, highlighting an important functional role for ASXL3 in PR-DUB mediated deubiquitination. Transcriptomes of ASXL3 patient and control fibroblasts were compared to investigate the impact of chromatin changes on transcriptional regulation. Out of 564 significantly differentially expressed genes (DEGs) in ASXL3 patient fibroblasts, 52% were upregulated and 48% downregulated. DEGs were enriched in molecular processes impacting transcriptional regulation, development and proliferation, consistent with the features of BRS. This is the first single gene disorder linked to defects in deubiquitination of H2AK119Ub1 and suggests an important role for dynamic regulation of H2A mono-ubiquitination in transcriptional regulation and the pathophysiology of BRS.

Photosensitivity and type I IFN responses in cutaneous lupus are driven by epidermal-derived interferon kappa.

OBJECTIVE: Skin inflammation and photosensitivity are common in patients with cutaneous lupus erythematosus (CLE) and systemic lupus erythematosus (SLE), yet little is known about the mechanisms that regulate these traits. Here we investigate the role of interferon kappa (IFN-κ) in regulation of type I interferon (IFN) and photosensitive responses and examine its dysregulation in lupus skin. METHODS: mRNA expression of type I IFN genes was analysed from microarray data of CLE lesions and healthy control skin. Similar expression in cultured primary keratinocytes, fibroblasts and endothelial cells was analysed via RNA-seq. IFNK knock-out (KO) keratinocytes were generated using CRISPR/Cas9. Keratinocytes stably overexpressing IFN-κ were created via G418 selection of transfected cells. IFN responses were assessed via phosphorylation of STAT1 and STAT2 and qRT-PCR for IFN-regulated genes. Ultraviolet B-mediated apoptosis was analysed via TUNEL staining. In vivo protein expression was assessed via immunofluorescent staining of normal and CLE lesional skin. RESULTS: IFNK is one of two type I IFNs significantly increased (1.5-fold change, false discovery rate (FDR) q<0.001) in lesional CLE skin. Gene ontology (GO) analysis showed that type I IFN responses were enriched (FDR=6.8×10-04) in keratinocytes not in fibroblast and endothelial cells, and this epithelial-derived IFN-κ is responsible for maintaining baseline type I IFN responses in healthy skin. Increased levels of IFN-κ, such as seen in SLE, amplify and accelerate responsiveness of epithelia to IFN-α and increase keratinocyte sensitivity to UV irradiation. Notably, KO of IFN-κ or inhibition of IFN signalling with baricitinib abrogates UVB-induced apoptosis. CONCLUSION: Collectively, our data identify IFN-κ as a critical IFN in CLE pathology via promotion of enhanced IFN responses and photosensitivity. IFN-κ is a potential novel target for UVB prophylaxis and CLE-directed therapy.

Genetic analysis of CHARGE syndrome identifies overlapping molecular biology.

PURPOSE: CHARGE syndrome is an autosomal-dominant, multiple congenital anomaly condition characterized by vision and hearing loss, congenital heart disease, and malformations of craniofacial and other structures. Pathogenic variants in CHD7, encoding adenosine triphosphate-dependent chromodomain helicase DNA binding protein 7, are present in the majority of affected individuals. However, no causal variant can be found in 5-30% (depending on the cohort) of individuals with a clinical diagnosis of CHARGE syndrome. METHODS: We performed whole-exome sequencing (WES) on 28 families from which at least one individual presented with features highly suggestive of CHARGE syndrome. RESULTS: Pathogenic variants in CHD7 were present in 15 of 28 individuals (53.6%), whereas 4 (14.3%) individuals had pathogenic variants in other genes (RERE, KMT2D, EP300, or PUF60). A variant of uncertain clinical significance in KDM6A was identified in one (3.5%) individual. The remaining eight (28.6%) individuals were not found to have pathogenic variants by WES. CONCLUSION: These results demonstrate that the phenotypic features of CHARGE syndrome overlap with multiple other rare single-gene syndromes. Additionally, they implicate a shared molecular pathology that disrupts epigenetic regulation of multiple-organ development.

Homozygous mutation in NUP107 leads to microcephaly with steroid-resistant nephrotic condition similar to Galloway-Mowat syndrome.

BACKGROUND: Microcephaly with nephrotic syndrome is a rare co-occurrence, constituting the Galloway-Mowat syndrome (GAMOS), caused by mutations in WDR73 (OMIM: 616144). However, not all patients harbour demonstrable WDR73 deleterious variants, suggesting that there are other yet unidentified factors contributing to GAMOS aetiology. METHODS: Autozygosity mapping and candidate analysis was used to identify deleterious variants in consanguineous families. Analysis of patient fibroblasts was used to study splicing and alterations in cellular function. RESULTS: In two consanguineous families with five affected individuals from Turkey with a GAMOS-like presentation, we identified a shared homozygous variant leading to partial exon 4 skipping in nucleoporin, 107-KD (NUP107). The founder mutation was associated with concomitant reduction in NUP107 protein and in the obligate binding partner NUP133 protein, as well as density of nuclear pores in patient cells. CONCLUSION: Recently, NUP107 was suggested as a candidate in a family with nephrotic syndrome and developmental delay. Other NUP107-reported cases had isolated renal phenotypes. With the addition of these individuals, we implicate an allele-specific critical role for NUP107 in the regulation of brain growth and a GAMOS-like presentation.

Quiescence enables unrestricted cell fate in naive embryonic stem cells.

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.

TREX tetramer disruption alters RNA processing necessary for corticogenesis in THOC6 Intellectual Disability Syndrome.

THOC6 variants are the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 is critical for mammalian Transcription Export complex (TREX) tetramer formation, which is composed of four six-subunit THO monomers. The TREX tetramer facilitates mammalian RNA processing, in addition to the nuclear mRNA export functions of the TREX dimer conserved through yeast. Human and mouse TIDS model systems revealed novel THOC6-dependent, species-specific TREX tetramer functions. Germline biallelic Thoc6 loss-of-function (LOF) variants result in mouse embryonic lethality. Biallelic THOC6 LOF variants reduce the binding affinity of ALYREF to THOC5 without affecting the protein expression of TREX members, implicating impaired TREX tetramer formation. Defects in RNA nuclear export functions were not detected in biallelic THOC6 LOF human neural cells. Instead, mis-splicing was detected in human and mouse neural tissue, revealing novel THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for key signaling pathways known to regulate the transition from proliferative to neurogenic divisions during human corticogenesis. Together, these findings implicate altered RNA processing in the developmental biology of TIDS neuropathology.

Biallelic variants in CSMD1 are implicated in a neurodevelopmental disorder with intellectual disability and variable cortical malformations.

CSMD1 (Cub and Sushi Multiple Domains 1) is a well-recognized regulator of the complement cascade, an important component of the innate immune response. CSMD1 is highly expressed in the central nervous system (CNS) where emergent functions of the complement pathway modulate neural development and synaptic activity. While a genetic risk factor for neuropsychiatric disorders, the role of CSMD1 in neurodevelopmental disorders is unclear. Through international variant sharing, we identified inherited biallelic CSMD1 variants in eight individuals from six families of diverse ancestry who present with global developmental delay, intellectual disability, microcephaly, and polymicrogyria. We modeled CSMD1 loss-of-function (LOF) pathogenesis in early-stage forebrain organoids differentiated from CSMD1 knockout human embryonic stem cells (hESCs). We show that CSMD1 is necessary for neuroepithelial cytoarchitecture and synchronous differentiation. In summary, we identified a critical role for CSMD1 in brain development and biallelic CSMD1 variants as the molecular basis of a previously undefined neurodevelopmental disorder.

De novo variants in GATAD2A in individuals with a neurodevelopmental disorder: GATAD2A-related neurodevelopmental disorder.

GATA zinc finger domain containing 2A (GATAD2A) is a subunit of the nucleosome remodeling and deacetylase (NuRD) complex. NuRD is known to regulate gene expression during neural development and other processes. The NuRD complex modulates chromatin status through histone deacetylation and ATP-dependent chromatin remodeling activities. Several neurodevelopmental disorders (NDDs) have been previously linked to variants in other components of NuRD's chromatin remodeling subcomplex (NuRDopathies). We identified five individuals with features of an NDD that possessed de novo autosomal dominant variants in GATAD2A. Core features in affected individuals include global developmental delay, structural brain defects, and craniofacial dysmorphology. These GATAD2A variants are predicted to affect protein dosage and/or interactions with other NuRD chromatin remodeling subunits. We provide evidence that a GATAD2A missense variant disrupts interactions of GATAD2A with CHD3, CHD4, and CHD5. Our findings expand the list of NuRDopathies and provide evidence that GATAD2A variants are the genetic basis of a previously uncharacterized developmental disorder.

Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome.

Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the "molar tooth sign" on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs.

Myofibrillar Structural Variability Underlies Contractile Function in Stem Cell-Derived Cardiomyocytes.

Disease modeling and pharmaceutical testing using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) requires accurate assessment of contractile function. Micropatterning iPSC-CMs on elastic substrates controls cell shape and alignment to enable contractile studies, but determinants of intrinsic variability in this system have been incompletely characterized. The objective of this study was to determine the impact of myofibrillar structure on contractile function in iPSC-CMs. Automated analysis of micropatterned iPSC-CMs labeled with a cell-permeant F-actin dye revealed that myofibrillar abundance is widely variable among iPSC-CMs and strongly correlates with contractile function. This variability is not reduced by subcloning from single iPSCs and is independent of the iPSC-CM purification method. Controlling for myofibrillar structure reduces false-positive findings related to batch effect and improves sensitivity for pharmacologic testing and disease modeling. This analysis provides compelling evidence that myofibrillar structure should be assessed concurrently in studies investigating contractile function in iPSC-CMs.

Modeling Bainbridge-Ropers Syndrome in Xenopus laevis Embryos.

The Additional sex combs-like (ASXL1-3) genes are linked to human neurodevelopmental disorders. The de novo truncating variants in ASXL1-3 proteins serve as the genetic basis for severe neurodevelopmental diseases such as Bohring-Opitz, Shashi-Pena, and Bainbridge-Ropers syndromes, respectively. The phenotypes of these syndromes are similar but not identical, and include dramatic craniofacial defects, microcephaly, developmental delay, and severe intellectual disability, with a loss of speech and language. Bainbridge-Ropers syndrome resulting from ASXL3 gene mutations also includes features of autism spectrum disorder. Human genomic studies also identified missense ASXL3 variants associated with autism spectrum disorder, but lacking more severe Bainbridge-Ropers syndromic features. While these findings strongly implicate ASXL3 in mammalian brain development, its functions are not clearly understood. ASXL3 protein is a component of the polycomb deubiquitinase complex that removes mono-ubiquitin from Histone H2A. Dynamic chromatin modifications play important roles in the specification of cell fates during early neural patterning and development. In this study, we utilize the frog, Xenopus laevis as a simpler and more accessible vertebrate neurodevelopmental model system to understand the embryological cause of Bainbridge-Ropers syndrome. We have found that ASXL3 protein knockdown during early embryo development highly perturbs neural cell fate specification, potentially resembling the Bainbridge-Ropers syndrome phenotype in humans. Thus, the frog embryo is a powerful tool for understanding the etiology of Bainbridge-Ropers syndrome in humans.

Recurrent 1q21.1 deletion syndrome: report on variable expression, nonpenetrance and review of literature.

The clinical phenotype of 1q21.1 microdeletion syndrome is highly heterogeneous. It is characterized by dysmorphic facial features, microcephaly, and developmental delay. Several congenital defects, including cardiac, ocular, skeletal anomalies, and psychiatric or behavioural abnormalities, have also been described. Here, we report on two siblings with substantial intrafamilial phenotypic variability carrying a heterozygous deletion of the 1q21.1 region spanning a known critical genomic area (~1.35 Mb). The microdeletion was inherited from the unaffected father. Patients described here show a spectrum of clinical features, a portion of which overlap with those previously reported in patients with 1q21.1 microdeletions. In addition, we review the clinical reports of 66 individuals with this condition. These findings extend and substantiate the current clinical understanding of recurrent copy number variations in the 1q21.1 region.