Epileptic encephalopathy (EE) refers to a clinically and genetically heterogeneous group of severe disorders characterized by seizures, abnormal interictal electro-encephalogram, psychomotor delay, and/or cognitive deterioration. We ascertained two multiplex families (including one consanguineous family) consistent with an autosomal-recessive inheritance pattern of EE. All seven affected individuals developed subclinical seizures as early as the first day of life, severe epileptic disease, and profound developmental delay with no facial dysmorphism. Given the similarity in clinical presentation in the two families, we hypothesized that the observed phenotype was due to mutations in the same gene, and we performed exome sequencing in three affected individuals. Analysis of rare variants in genes consistent with an autosomal-recessive mode of inheritance led to identification of mutations in SLC13A5, which encodes the cytoplasmic sodium-dependent citrate carrier, notably expressed in neurons. Disease association was confirmed by cosegregation analysis in additional family members. Screening of 68 additional unrelated individuals with early-onset epileptic encephalopathy for SLC13A5 mutations led to identification of one additional subject with compound heterozygous mutations of SLC13A5 and a similar clinical presentation as the index subjects. Mutations affected key residues for sodium binding, which is critical for citrate transport. These findings underline the value of careful clinical characterization for genetic investigations in highly heterogeneous conditions such as EE and further highlight the role of citrate metabolism in epilepsy.

Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in NEDD4L mapping to the HECT domain of the encoded E3 ubiquitin ligase lead to PNH associated with toe syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed sensitivity of PNH-associated mutants to proteasome degradation. Moreover, an in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these data provide insights into the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development.

Intellectual disability (ID) affects approximately 1%-3% of humans with a gender bias toward males. Previous studies have identified mutations in more than 100 genes on the X chromosome in males with ID, but there is less evidence for de novo mutations on the X chromosome causing ID in females. In this study we present 35 unique deleterious de novo mutations in DDX3X identified by whole exome sequencing in 38 females with ID and various other features including hypotonia, movement disorders, behavior problems, corpus callosum hypoplasia, and epilepsy. Based on our findings, mutations in DDX3X are one of the more common causes of ID, accounting for 1%-3% of unexplained ID in females. Although no de novo DDX3X mutations were identified in males, we present three families with segregating missense mutations in DDX3X, suggestive of an X-linked recessive inheritance pattern. In these families, all males with the DDX3X variant had ID, whereas carrier females were unaffected. To explore the pathogenic mechanisms accounting for the differences in disease transmission and phenotype between affected females and affected males with DDX3X missense variants, we used canonical Wnt defects in zebrafish as a surrogate measure of DDX3X function in vivo. We demonstrate a consistent loss-of-function effect of all tested de novo mutations on the Wnt pathway, and we further show a differential effect by gender. The differential activity possibly reflects a dose-dependent effect of DDX3X expression in the context of functional mosaic females versus one-copy males, which reflects the complex biological nature of DDX3X mutations.

SON is a key component of the spliceosomal complex and a critical mediator of constitutive and alternative splicing. Additionally, SON has been shown to influence cell-cycle progression, genomic integrity, and maintenance of pluripotency in stem cell populations. The clear functional relevance of SON in coordinating essential cellular processes and its presence in diverse human tissues suggests that intact SON might be crucial for normal growth and development. However, the phenotypic effects of deleterious germline variants in SON have not been clearly defined. Herein, we describe seven unrelated individuals with de novo variants in SON and propose that deleterious variants in SON are associated with a severe multisystem disorder characterized by developmental delay, persistent feeding difficulties, and congenital malformations, including brain anomalies.

Cantú syndrome is a rare disorder characterized by congenital hypertrichosis, neonatal macrosomia, a distinct osteochondrodysplasia, and cardiomegaly. Using an exome-sequencing approach applied to one proband-parent trio and three unrelated single cases, we identified heterozygous mutations in ABCC9 in all probands. With the inclusion of the remaining cohort of ten individuals with Cantú syndrome, a total of eleven mutations in ABCC9 were found. The de novo occurrence in all six simplex cases in our cohort substantiates the presence of a dominant disease mechanism. All mutations were missense, and several mutations affect Arg1154. This mutation hot spot lies within the second type 1 transmembrane region of this ATP-binding cassette transporter protein, which may suggest an activating mutation. ABCC9 encodes the sulfonylurea receptor (SUR) that forms ATP-sensitive potassium channels (K(ATP) channels) originally shown in cardiac, skeletal, and smooth muscle. Previously, loss-of-function mutations in this gene have been associated with idiopathic dilated cardiomyopathy type 10 (CMD10). These findings identify the genetic basis of Cantú syndrome and suggest that this is a new member of the potassium channelopathies.

RORα, the RAR-related orphan nuclear receptor alpha, is essential for cerebellar development. The spontaneous mutant mouse staggerer, with an ataxic gait caused by neurodegeneration of cerebellar Purkinje cells, was discovered two decades ago to result from homozygous intragenic Rora deletions. However, RORA mutations were hitherto undocumented in humans. Through a multi-centric collaboration, we identified three copy-number variant deletions (two de novo and one dominantly inherited in three generations), one de novo disrupting duplication, and nine de novo point mutations (three truncating, one canonical splice site, and five missense mutations) involving RORA in 16 individuals from 13 families with variable neurodevelopmental delay and intellectual disability (ID)-associated autistic features, cerebellar ataxia, and epilepsy. Consistent with the human and mouse data, disruption of the D. rerio ortholog, roraa, causes significant reduction in the size of the developing cerebellum. Systematic in vivo complementation studies showed that, whereas wild-type human RORA mRNA could complement the cerebellar pathology, missense variants had two distinct pathogenic mechanisms of either haploinsufficiency or a dominant toxic effect according to their localization in the ligand-binding or DNA-binding domains, respectively. This dichotomous direction of effect is likely relevant to the phenotype in humans: individuals with loss-of-function variants leading to haploinsufficiency show ID with autistic features, while individuals with de novo dominant toxic variants present with ID, ataxia, and cerebellar atrophy. Our combined genetic and functional data highlight the complex mutational landscape at the human RORA locus and suggest that dual mutational effects likely determine phenotypic outcome.

The 15q11.2 deletion is frequently identified in the neurodevelopmental clinic. Case-control studies have associated the 15q11.2 deletion with neurodevelopmental disorders, and clinical case series have attempted to delineate a microdeletion syndrome with considerable phenotypic variability. The literature on this deletion is extensive and confusing, which is a challenge for genetic counselling. The aim of this study was to estimate the effect size of the 15q11.2 deletion and quantify its contribution to neurodevelopmental disorders.

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Variants in genes encoding ribosomal proteins have thus far been associated with Diamond-Blackfan anemia, a rare inherited bone marrow failure, and isolated congenital asplenia. Here, we report one de novo missense variant and three de novo splice variants in RPL13, which encodes ribosomal protein RPL13 (also called eL13), in four unrelated individuals with a rare bone dysplasia causing severe short stature. The three splice variants (c.477+1G>T, c.477+1G>A, and c.477+2 T>C) result in partial intron retention, which leads to an 18-amino acid insertion. In contrast to observations from Diamond-Blackfan anemia, we detected no evidence of significant pre-rRNA processing disturbance in cells derived from two affected individuals. Consistently, we showed that the insertion-containing protein is stably expressed and incorporated into 60S subunits similar to the wild-type protein. Erythroid proliferation in culture and ribosome profile on sucrose gradient are modified, suggesting a change in translation dynamics. We also provide evidence that RPL13 is present at high levels in chondrocytes and osteoblasts in mouse growth plates. Taken together, we show that the identified RPL13 variants cause a human ribosomopathy defined by a rare skeletal dysplasia, and we highlight the role of this ribosomal protein in bone development.

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

SOX6 belongs to a family of 20 SRY-related HMG-box-containing (SOX) genes that encode transcription factors controlling cell fate and differentiation in many developmental and adult processes. For SOX6, these processes include, but are not limited to, neurogenesis and skeletogenesis. Variants in half of the SOX genes have been shown to cause severe developmental and adult syndromes, referred to as SOXopathies. We here provide evidence that SOX6 variants also cause a SOXopathy. Using clinical and genetic data, we identify 19 individuals harboring various types of SOX6 alterations and exhibiting developmental delay and/or intellectual disability; the individuals are from 17 unrelated families. Additional, inconstant features include attention-deficit/hyperactivity disorder (ADHD), autism, mild facial dysmorphism, craniosynostosis, and multiple osteochondromas. All variants are heterozygous. Fourteen are de novo, one is inherited from a mosaic father, and four offspring from two families have a paternally inherited variant. Intragenic microdeletions, balanced structural rearrangements, frameshifts, and nonsense variants are predicted to inactivate the SOX6 variant allele. Four missense variants occur in residues and protein regions highly conserved evolutionarily. These variants are not detected in the gnomAD control cohort, and the amino acid substitutions are predicted to be damaging. Two of these variants are located in the HMG domain and abolish SOX6 transcriptional activity in vitro. No clear genotype-phenotype correlations are found. Taken together, these findings concur that SOX6 haploinsufficiency leads to a neurodevelopmental SOXopathy that often includes ADHD and abnormal skeletal and other features.

The neuro-oncological ventral antigen 2 (NOVA2) protein is a major factor regulating neuron-specific alternative splicing (AS), previously associated with an acquired neurologic condition, the paraneoplastic opsoclonus-myoclonus ataxia (POMA). We report here six individuals with de novo frameshift variants in NOVA2 affected with a severe neurodevelopmental disorder characterized by intellectual disability (ID), motor and speech delay, autistic features, hypotonia, feeding difficulties, spasticity or ataxic gait, and abnormal brain MRI. The six variants lead to the same reading frame, adding a common proline rich C-terminal part instead of the last KH RNA binding domain. We detected 41 genes differentially spliced after NOVA2 downregulation in human neural cells. The NOVA2 variant protein shows decreased ability to bind target RNA sequences and to regulate target AS events. It also fails to complement the effect on neurite outgrowth induced by NOVA2 downregulation in vitro and to rescue alterations of retinotectal axonal pathfinding induced by loss of NOVA2 ortholog in zebrafish. Our results suggest a partial loss-of-function mechanism rather than a full heterozygous loss-of-function, although a specific contribution of the novel C-terminal extension cannot be excluded.

Kinesin-2 enables ciliary assembly and maintenance as an anterograde intraflagellar transport (IFT) motor. Molecular motor activity is driven by a heterotrimeric complex comprised of KIF3A and KIF3B or KIF3C plus one non-motor subunit, KIFAP3. Using exome sequencing, we identified heterozygous KIF3B variants in two unrelated families with hallmark ciliopathy phenotypes. In the first family, the proband presents with hepatic fibrosis, retinitis pigmentosa, and postaxial polydactyly; he harbors a de novo c.748G>C (p.Glu250Gln) variant affecting the kinesin motor domain encoded by KIF3B. The second family is a six-generation pedigree affected predominantly by retinitis pigmentosa. Affected individuals carry a heterozygous c.1568T>C (p.Leu523Pro) KIF3B variant segregating in an autosomal-dominant pattern. We observed a significant increase in primary cilia length in vitro in the context of either of the two mutations while variant KIF3B proteins retained stability indistinguishable from wild type. Furthermore, we tested the effects of KIF3B mutant mRNA expression in the developing zebrafish retina. In the presence of either missense variant, rhodopsin was sequestered to the photoreceptor rod inner segment layer with a concomitant increase in photoreceptor cilia length. Notably, impaired rhodopsin trafficking is also characteristic of recessive KIF3B models as exemplified by an early-onset, autosomal-recessive, progressive retinal degeneration in Bengal cats; we identified a c.1000G>A (p.Ala334Thr) KIF3B variant by genome-wide association study and whole-genome sequencing. Together, our genetic, cell-based, and in vivo modeling data delineate an autosomal-dominant syndromic retinal ciliopathy in humans and suggest that multiple KIF3B pathomechanisms can impair kinesin-driven ciliary transport in the photoreceptor.

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.

Most genes associated with neurodevelopmental disorders (NDDs) were identified with an excess of de novo mutations (DNMs) but the significance in case-control mutation burden analysis is unestablished. Here, we sequence 63 genes in 16,294 NDD cases and an additional 62 genes in 6,211 NDD cases. By combining these with published data, we assess a total of 125 genes in over 16,000 NDD cases and compare the mutation burden to nonpsychiatric controls from ExAC. We identify 48 genes (25 newly reported) showing significant burden of ultra-rare (MAF < 0.01%) gene-disruptive mutations (FDR 5%), six of which reach family-wise error rate (FWER) significance (p < 1.25E-06). Among these 125 targeted genes, we also reevaluate DNM excess in 17,426 NDD trios with 6,499 new autism trios. We identify 90 genes enriched for DNMs (FDR 5%; e.g., GABRG2 and UIMC1); of which, 61 reach FWER significance (p < 3.64E-07; e.g., CASZ1). In addition to doubling the number of patients for many NDD risk genes, we present phenotype-genotype correlations for seven risk genes (CTCF, HNRNPU, KCNQ3, ZBTB18, TCF12, SPEN, and LEO1) based on this large-scale targeted sequencing effort.

Congenital disorders of glycosylation (CDG) constitute an ever-growing group of genetic diseases affecting the glycosylation of proteins. CDG individuals usually present with severe multisystem disorders. MAN1B1-CDG is a CDG with nonspecific clinical symptoms such as intellectual deficiency and developmental delay. Although up to 40 affected individuals were described so far, its final diagnosis is not straightforward using common biochemical methods due to the trace-level accumulation of defective glycan structures. In this study, we present three unreported MAN1B1-CDG individuals and propose a decision tree to reach diagnosis using a panel of techniques ranging from exome sequencing to gel electrophoresis and mass spectrometry. The occurrence of MAN1B1-CDG in patients showing unexplained intellectual disability and development delay, as well as a particular transferrin glycosylation profile, can be ascertained notably using matrix assisted laser desorption/ionization - time of flight (MALDI-TOF) mass spectrometry analysis of endo-β-acetylglucosaminidase H-released serum N-glycans. In addition to reporting new pathogenic variants and additional clinical signs such as hypersialorrhea, we highlight particular biochemical features of MAN1B1-CDG with potential glycoprotein-specific glycosylation defects.

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability.

The MYH7 c.5135G > A p.(Arg1712Gln) variant has been identified in several patients worldwide and is classified as pathogenic in the ClinVar database. We aimed to delineate its associated phenotype and evaluate a potential founder effect.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities.