Genetic and phenotypic landscape of pediatric-onset epilepsy in 142 Indian families: Counseling and therapeutic implications.

The application of genomic technologies has led to unraveling of the complex genetic landscape of disorders of epilepsy, gaining insights into their underlying disease mechanisms, aiding precision medicine, and providing informed genetic counseling. We herein present the phenotypic and genotypic insights from 142 Indian families with epilepsy with or without comorbidities. Based on the electroclinical findings, epilepsy syndrome diagnosis could be made in 44% (63/142) of the families adopting the latest proposal for the classification by the ILAE task force (2022). Of these, 95% (60/63) of the families exhibited syndromes with developmental epileptic encephalopathy or progressive neurological deterioration. A definitive molecular diagnosis was achieved in 74 of 142 (52%) families. Infantile-onset epilepsy was noted in 81% of these families (61/74). Fifty-five monogenic, four chromosomal, and one imprinting disorder were identified in 74 families. The genetic variants included 65 (96%) single-nucleotide variants/small insertion-deletions, 1 (2%) copy-number variant, and 1 (2%) triplet-repeat expansion in 53 epilepsy-associated genes causing monogenic disorders. Of these, 35 (52%) variants were novel. Therapeutic implications were noted in 51% of families (38/74) with definitive diagnosis. Forty-one out of 66 families with monogenic disorders exhibited autosomal recessive and inherited autosomal dominant disorders with high risk of recurrence.

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.

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.

De novo variants underlying monogenic syndromes with intellectual disability in a neurodevelopmental cohort from India.

The contribution of de novo variants as a cause of intellectual disability (ID) is well established in several cohorts reported from the developed world. However, the genetic landscape as well as the appropriate testing strategies for identification of de novo variants of these disorders remain largely unknown in low-and middle-income countries like India. In this study, we delineate the clinical and genotypic spectrum of 54 families (55 individuals) with syndromic ID harboring rare de novo variants. We also emphasize on the effectiveness of singleton exome sequencing as a valuable tool for diagnosing these disorders in resource limited settings. Overall, 46 distinct disorders were identified encompassing 46 genes with 51 single-nucleotide variants and/or indels and two copy-number variants. Pathogenic variants were identified in CREBBP, TSC2, KMT2D, MECP2, IDS, NIPBL, NSD1, RIT1, SOX10, BRWD3, FOXG1, BCL11A, KDM6B, KDM5C, SETD5, QRICH1, DCX, SMARCD1, ASXL1, ASXL3, AKT3, FBN2, TCF12, WASF1, BRAF, SMARCA4, SMARCA2, TUBG1, KMT2A, CTNNB1, DLG4, MEIS2, GATAD2B, FBXW7, ANKRD11, ARID1B, DYNC1H1, HIVEP2, NEXMIF, ZBTB18, SETD1B, DYRK1A, SRCAP, CASK, L1CAM, and KRAS. Twenty-four of these monogenic disorders have not been previously reported in the Indian population. Notably, 39 out of 53 (74%) disease-causing variants are novel. These variants were identified in the genes mainly encoding transcriptional and chromatin regulators, serine threonine kinases, lysosomal enzymes, molecular motors, synaptic proteins, neuronal migration machinery, adhesion molecules, structural proteins and signaling molecules.

Mechanisms of mRNA processing defects in inherited THOC6 intellectual disability syndrome.

THOC6 is the genetic basis of autosomal recessive THOC6 Intellectual Disability Syndrome (TIDS). THOC6 facilitates the formation of the Transcription Export complex (TREX) tetramer, composed of four THO monomers. The TREX tetramer supports mammalian mRNA processing that is distinct from yeast TREX dimer functions. Human and mouse TIDS model systems allow novel THOC6-dependent TREX tetramer functions to be investigated. Biallelic loss-of-functon(LOF) THOC6 variants do not influence the expression and localization of TREX members in human cells, but our data suggests reduced binding affinity of ALYREF. Impairment of TREX nuclear export functions were not detected in cells with biallelic THOC6 LOF. Instead, mRNA mis-splicing was observed in human and mouse neural tissue, revealing novel insights into THOC6-mediated TREX coordination of mRNA processing. We demonstrate that THOC6 is required for regulation of key signaling pathways in human corticogenesis that dictate the transition from proliferative to neurogenic divisions that may inform TIDS neuropathology.

Further validation of craniosynostosis as a part of phenotypic spectrum of BCL11B-related BAFopathy.

Heterozygous disease-causing variants in BCL11B are the basis of a rare neurodevelopmental syndrome with craniofacial and immunological involvement. Isolated craniosynostosis, without systemic or immunological findings, has been reported in one of the 17 individuals reported with this disorder till date. We report three additional individuals harboring de novo heterozygous frameshift variants, all lying in the exon 4 of BCL11B. All three individuals presented with the common findings of this disorder i.e. developmental delay, recurrent infections with immunologic abnormalities and facial dysmorphism. Notably, craniosynostosis of variable degree was seen in all three individuals. We, thus add to the evolving genotypes and phenotypes of BCL11B-related BAFopathy and also review the clinical, genomic spectrum along with the underlying disease mechanisms of this 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.

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.

Molecular and functional heterogeneity in dorsal and ventral oligodendrocyte progenitor cells of the mouse forebrain in response to DNA damage.

In the developing mouse forebrain, temporally distinct waves of oligodendrocyte progenitor cells (OPCs) arise from different germinal zones and eventually populate either dorsal or ventral regions, where they present as transcriptionally and functionally equivalent cells. Despite that, developmental heterogeneity influences adult OPC responses upon demyelination. Here we show that accumulation of DNA damage due to ablation of citron-kinase or cisplatin treatment cell-autonomously disrupts OPC fate, resulting in cell death and senescence in the dorsal and ventral subsets, respectively. Such alternative fates are associated with distinct developmental origins of OPCs, and with a different activation of NRF2-mediated anti-oxidant responses. These data indicate that, upon injury, dorsal and ventral OPC subsets show functional and molecular diversity that can make them differentially vulnerable to pathological conditions associated with DNA damage.

Multilocus disease-causing genomic variations for Mendelian disorders: role of systematic phenotyping and implications on genetic counselling.

Multilocus disease-causing genomic variations (MGVs) and multiple genetic diagnoses (MGDs) are increasingly being recognised in individuals and families with Mendelian disorders. This can be mainly attributed to the widespread use of genomic tests for the evaluation of these disorders. We conducted a retrospective study of families evaluated over the last 6 years at our centre to identify families with MGVs and MGDs. MGVs were observed in fourteen families. We observed five different consequences: (i) individuals with MGVs presenting as blended phenotypes (ii) individuals with MGVs presenting with distinct phenotypes (iii) individuals with MGVs with age-dependent penetrance (iv) individuals with MGVs with one phenotype obscured by another more predominant phenotype (v) two distinct phenotypes in different individuals in families with MGVs. Consanguinity was present in eight (8/14, 57.1%) of them. Thirteen families had two Mendelian disorders and one had three Mendelian disorders. The risk of recurrence of one or more conditions in these families ranged from 25% to 75%. Our findings underline the importance of the role of a clinical geneticist in systematic phenotyping, challenges in genetic counselling and risk estimation in families with MGVs and MGDs, especially in highly inbred populations.

Clinical and genetic spectrum of 104 Indian families with central nervous system white matter abnormalities.

Genetic disorders with predominant central nervous system white matter abnormalities (CNS WMAs), also called leukodystrophies, are heterogeneous entities. We ascertained 117 individuals with CNS WMAs from 104 unrelated families. Targeted genetic testing was carried out in 16 families and 13 of them received a diagnosis. Chromosomal microarray (CMA) was performed for three families and one received a diagnosis. Mendeliome sequencing was used for testing 11 families and all received a diagnosis. Whole exome sequencing (WES) was performed in 80 families and was diagnostic in 52 (65%). Singleton WES was diagnostic for 50/75 (66.67%) families. Overall, genetic diagnoses were obtained in 77 families (74.03%). Twenty-two of 47 distinct disorders observed in this cohort have not been reported in Indian individuals previously. Notably, disorders of nuclear mitochondrial pathology were most frequent (9 disorders in 20 families). Thirty-seven of 75 (49.33%) disease-causing variants are novel. To sum up, the present cohort describes the phenotypic and genotypic spectrum of genetic disorders with CNS WMAs in our population. It demonstrates WES, especially singleton WES, as an efficient tool in the diagnosis of these heterogeneous entities. It also highlights possible founder events and recurrent disease-causing variants in our population and their implications on the testing strategy.

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.

Bi-allelic missense variant, p.Ser35Leu in EXOSC1 is associated with pontocerebellar hypoplasia.

RNA exosome is a highly conserved ribonuclease complex essential for RNA processing and degradation. Bi-allelic variants in exosome subunits EXOSC3, EXOSC8 and EXOSC9 have been reported to cause pontocerebellar hypoplasia type 1B, type 1C and type 1D, respectively, while those in EXOSC2 cause short stature, hearing loss, retinitis pigmentosa and distinctive facies. We ascertained an 8-months-old male with developmental delay, microcephaly, subtle dysmorphism and hypotonia. Pontocerebellar hypoplasia and delayed myelination were noted on neuroimaging. A similarly affected elder sibling succumbed at the age of 4-years 6-months. Chromosomal microarray returned normal results. Exome sequencing revealed a homozygous missense variant, c.104C > T p.(Ser35Leu) in EXOSC1 (NM_016046.5) as the possible candidate. In silico mutagenesis revealed loss of a polar contact with neighboring Leu37 residue. Quantitative real-time PCR indicated no appreciable differences in EXOSC1 transcript levels. Immunoblotting and blue native PAGE revealed reduction in the EXOSC1 protein levels and EXO9 complex in the proband, respectively. We herein report an individual with the bi-allelic variant c.104C>T p.(Ser35Leu) in EXOSC1 and clinical features of pontocerebellar hypoplasia type 1. Immunoblotting and blue native PAGE provide evidence for the pathogenicity of the variant. Thus, we propose EXOSC1 as a novel candidate gene for pontocerebellar hypoplasia.

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.

CHD7 promotes neural progenitor differentiation in embryonic stem cells via altered chromatin accessibility and nascent gene expression.

CHARGE syndrome, a rare multiple congenital anomaly condition, is caused by haploinsufficiency of the chromatin remodeling protein gene CHD7 (Chromodomain helicase DNA binding protein 7). Brain abnormalities and intellectual disability are commonly observed in individuals with CHARGE, and neuronal differentiation is reduced in CHARGE patient-derived iPSCs and conditional knockout mouse brains. However, the mechanisms of CHD7 function in nervous system development are not well understood. In this study, we asked whether CHD7 promotes gene transcription in neural progenitor cells via changes in chromatin accessibility. We used Chd7 null embryonic stem cells (ESCs) derived from Chd7 mutant mouse blastocysts as a tool to investigate roles of CHD7 in neuronal and glial differentiation. Loss of Chd7 significantly reduced neuronal and glial differentiation. Sholl analysis showed that loss of Chd7 impaired neuronal complexity and neurite length in differentiated neurons. Genome-wide studies demonstrated that loss of Chd7 leads to modified chromatin accessibility (ATAC-seq) and differential nascent expression (Bru-Seq) of neural-specific genes. These results suggest that CHD7 acts preferentially to alter chromatin accessibility of key genes during the transition of NPCs to neurons to promote differentiation. Our results form a basis for understanding the cell stage-specific roles for CHD7-mediated chromatin remodeling during cell lineage acquisition.

Histone Acetyltransferase MOF Blocks Acquisition of Quiescence in Ground-State ESCs through Activating Fatty Acid Oxidation.

Self-renewing embryonic stem cells (ESCs) respond to environmental cues by exiting pluripotency or entering a quiescent state. The molecular basis underlying this fate choice remains unclear. Here, we show that histone acetyltransferase MOF plays a critical role in this process through directly activating fatty acid oxidation (FAO) in the ground-state ESCs. We further show that the ground-state ESCs particularly rely on elevated FAO for oxidative phosphorylation (OXPHOS) and energy production. Mof deletion or FAO inhibition induces bona fide quiescent ground-state ESCs with an intact core pluripotency network and transcriptome signatures akin to the diapaused epiblasts in vivo. Mechanistically, MOF/FAO inhibition acts through reducing mitochondrial respiration (i.e., OXPHOS), which in turn triggers reversible pluripotent quiescence specifically in the ground-state ESCs. The inhibition of FAO/OXPHOS also induces quiescence in naive human ESCs. Our study suggests a general function of the MOF/FAO/OXPHOS axis in regulating cell fate determination in stem cells.

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.

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.