Mono-allelic KCNB2 variants lead to a neurodevelopmental syndrome caused by altered channel inactivation.

Ion channels mediate voltage fluxes or action potentials that are central to the functioning of excitable cells such as neurons. The KCNB family of voltage-gated potassium channels (Kv) consists of two members (KCNB1 and KCNB2) encoded by KCNB1 and KCNB2, respectively. These channels are major contributors to delayed rectifier potassium currents arising from the neuronal soma which modulate overall excitability of neurons. In this study, we identified several mono-allelic pathogenic missense variants in KCNB2, in individuals with a neurodevelopmental syndrome with epilepsy and autism in some individuals. Recurrent dysmorphisms included a broad forehead, synophrys, and digital anomalies. Additionally, we selected three variants where genetic transmission has not been assessed, from two epilepsy studies, for inclusion in our experiments. We characterized channel properties of these variants by expressing them in oocytes of Xenopus laevis and conducting cut-open oocyte voltage clamp electrophysiology. Our datasets indicate no significant change in absolute conductance and conductance-voltage relationships of most disease variants as compared to wild type (WT), when expressed either alone or co-expressed with WT-KCNB2. However, variants c.1141A>G (p.Thr381Ala) and c.641C>T (p.Thr214Met) show complete abrogation of currents when expressed alone with the former exhibiting a left shift in activation midpoint when expressed alone or with WT-KCNB2. The variants we studied, nevertheless, show collective features of increased inactivation shifted to hyperpolarized potentials. We suggest that the effects of the variants on channel inactivation result in hyper-excitability of neurons, which contributes to disease manifestations.

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features.

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.

Bi-allelic genetic variants in the translational GTPases GTPBP1 and GTPBP2 cause a distinct identical neurodevelopmental syndrome.

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.

Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity.

Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two de novo missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary ß subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.

Biallelic loss-of-function variants of SLC12A9 cause lysosome dysfunction and a syndromic neurodevelopmental disorder.

PURPOSE: Pathogenic variants of FIG4 generate enlarged lysosomes and neurological and developmental disorders. To identify additional genes regulating lysosomal volume, we carried out a genome-wide activation screen to detect suppression of enlarged lysosomes in FIG4-/- cells. METHODS: The CRISPR-a gene activation screen utilized sgRNAs from the promoters of protein-coding genes. Fluorescence-activated cell sorting separated cells with correction of the enlarged lysosomes from uncorrected cells. Patient variants of SLC12A9 were identified by exome or genome sequencing and studied by segregation analysis and clinical characterization. RESULTS: Overexpression of SLC12A9, a solute co-transporter, corrected lysosomal swelling in FIG4-/- cells. SLC12A9 (NP_064631.2) colocalized with LAMP2 at the lysosome membrane. Biallelic variants of SLC12A9 were identified in 3 unrelated probands with neurodevelopmental disorders. Common features included intellectual disability, skeletal and brain structural abnormalities, congenital heart defects, and hypopigmented hair. Patient 1 was homozygous for nonsense variant p.(Arg615∗), patient 2 was compound heterozygous for p.(Ser109Lysfs∗20) and a large deletion, and proband 3 was compound heterozygous for p.(Glu290Glyfs∗36) and p.(Asn552Lys). Fibroblasts from proband 1 contained enlarged lysosomes that were corrected by wild-type SLC12A9 cDNA. Patient variant p.(Asn552Lys) failed to correct the lysosomal defect. CONCLUSION: Impaired function of SLC12A9 results in enlarged lysosomes and a recessive disorder with a recognizable neurodevelopmental phenotype.

Biallelic loss-of-function variants in CACHD1 cause a novel neurodevelopmental syndrome with facial dysmorphism and multisystem congenital abnormalities.

PURPOSE: We established the genetic etiology of a syndromic neurodevelopmental condition characterized by variable cognitive impairment, recognizable facial dysmorphism, and a constellation of extra-neurological manifestations. METHODS: We performed phenotypic characterization of 6 participants from 4 unrelated families presenting with a neurodevelopmental syndrome and used exome sequencing to investigate the underlying genetic cause. To probe relevance to the neurodevelopmental phenotype and craniofacial dysmorphism, we established two- and three-dimensional human stem cell-derived neural models and generated a stable cachd1 zebrafish mutant on a transgenic cartilage reporter line. RESULTS: Affected individuals showed mild cognitive impairment, dysmorphism featuring oculo-auriculo abnormalities, and developmental defects involving genitourinary and digestive tracts. Exome sequencing revealed biallelic putative loss-of-function variants in CACHD1 segregating with disease in all pedigrees. RNA sequencing in CACHD1-depleted neural progenitors revealed abnormal expression of genes with key roles in Wnt signaling, neurodevelopment, and organ morphogenesis. CACHD1 depletion in neural progenitors resulted in reduced percentages of post-mitotic neurons and enlargement of 3D neurospheres. Homozygous cachd1 mutant larvae showed mandibular patterning defects mimicking human facial dysmorphism. CONCLUSION: Our findings support the role of loss-of-function variants in CACHD1 as the cause of a rare neurodevelopmental syndrome with facial dysmorphism and multisystem abnormalities.

Defining the Genetic Landscape of Congenital Mirror Movements in 80 Affected Individuals.

BACKGROUND: Congenital mirror movements (CMM) is a rare neurodevelopmental disorder characterized by involuntary movements from one side of the body that mirror voluntary movements on the opposite side. To date, five genes have been associated with CMM, namely DCC, RAD51, NTN1, ARHGEF7, and DNAL4. OBJECTIVE: The aim of this study is to characterize the genetic landscape of CMM in a large group of 80 affected individuals. METHODS: We screened 80 individuals with CMM from 43 families for pathogenic variants in CMM genes. In large CMM families, we tested for presence of pathogenic variants in multiple affected and unaffected individuals. In addition, we evaluated the impact of three missense DCC variants on binding between DCC and Netrin-1 in vitro. RESULTS: Causal pathogenic/likely pathogenic variants were found in 35% of probands overall, and 70% with familial CMM. The most common causal gene was DCC, responsible for 28% of CMM probands and 80% of solved cases. RAD51, NTN1, and ARHGEF7 were rare causes of CMM, responsible for 2% each. Penetrance of CMM in DCC pathogenic variant carriers was 68% and higher in males than females (74% vs. 54%). The three tested missense variants (p.Ile164Thr; p.Asn176Ser; and p.Arg1343His) bind Netrin-1 similarly to wild type DCC. CONCLUSIONS: A genetic etiology can be identified in one third of CMM individuals, with DCC being the most common gene involved. Two thirds of CMM individuals were unsolved, highlighting that CMM is genetically heterogeneous and other CMM genes are yet to be discovered. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Clinical and molecular characterization of patients with YWHAG-related epilepsy.

OBJECTIVE: YWHAG variant alleles have been associated with a rare disease trait whose clinical synopsis includes an early onset epileptic encephalopathy with predominantly myoclonic seizures, developmental delay/intellectual disability, and facial dysmorphisms. Through description of a large cohort, which doubles the number of reported patients, we further delineate the spectrum of YWHAG-related epilepsy. METHODS: We included in this study 24 patients, 21 new and three previously described, with pathogenic/likely pathogenic variants in YWHAG. We extended the analysis of clinical, electroencephalographic, brain magnetic resonance imaging, and molecular genetic information to 24 previously published patients. RESULTS: The phenotypic spectrum of YWHAG-related disorders ranges from mild developmental delay to developmental and epileptic encephalopathy (DEE). Epilepsy onset is in the first 2 years of life. Seizure freedom can be achieved in half of the patients (13/24, 54%). Intellectual disability (23/24, 96%), behavioral disorders (18/24, 75%), neurological signs (13/24, 54%), and dysmorphisms (6/24, 25%) are common. A genotype-phenotype correlation emerged, as DEE is more represented in patients with missense variants located in the ligand-binding domain than in those with truncating or missense variants in other domains (90% vs. 19%, p < .001). SIGNIFICANCE: This study suggests that pathogenic YWHAG variants cause a wide range of clinical presentations with variable severity, ranging from mild developmental delay to DEE. In this allelic series, a genotype-phenotype correlation begins to emerge, potentially providing prognostic information for clinical management and genetic counseling.

Genotype-phenotype correlation in contactin-associated protein-like 2 (CNTNAP-2) developmental disorder.

Contactin-associated protein-like 2 (CNTNAP2) gene encodes for CASPR2, a presynaptic type 1 transmembrane protein, involved in cell-cell adhesion and synaptic interactions. Biallelic CNTNAP2 loss has been associated with "Pitt-Hopkins-like syndrome-1" (MIM#610042), while the pathogenic role of heterozygous variants remains controversial. We report 22 novel patients harboring mono- (n = 2) and bi-allelic (n = 20) CNTNAP2 variants and carried out a literature review to characterize the genotype-phenotype correlation. Patients (M:F 14:8) were aged between 3 and 19 years and affected by global developmental delay (GDD) (n = 21), moderate to profound intellectual disability (n = 17) and epilepsy (n = 21). Seizures mainly started in the first two years of life (median 22.5 months). Antiseizure medications were successful in controlling the seizures in about two-thirds of the patients. Autism spectrum disorder (ASD) and/or other neuropsychiatric comorbidities were present in nine patients (40.9%). Nonspecific midline brain anomalies were noted in most patients while focal signal abnormalities in the temporal lobes were noted in three subjects. Genotype-phenotype correlation was performed by also including 50 previously published patients (15 mono- and 35 bi-allelic variants). Overall, GDD (p < 0.0001), epilepsy (p < 0.0001), hyporeflexia (p = 0.012), ASD (p = 0.009), language impairment (p = 0.020) and severe cognitive impairment (p = 0.031) were significantly associated with the presence of biallelic versus monoallelic variants. We have defined the main features associated with biallelic CNTNAP2 variants, as severe cognitive impairment, epilepsy and behavioral abnormalities. We propose CASPR2-deficiency neurodevelopmental disorder as an exclusively recessive disease while the contribution of heterozygous variants is less likely to follow an autosomal dominant inheritance pattern.

Clinical, neuroradiological, and molecular characterization of mitochondrial threonyl-tRNA-synthetase (TARS2)-related disorder.

PURPOSE: Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype but with limited neuroradiological data and insufficient evidence for causality of the variants. METHODS: Exome or genome sequencing was carried out in 15 families. Clinical and neuroradiological evaluation was performed for all affected individuals, including review of 10 previously reported individuals. The pathogenicity of TARS2 variants was evaluated using in vitro assays and a zebrafish model. RESULTS: We report 18 new individuals harboring biallelic TARS2 variants. Phenotypically, these individuals show developmental delay/intellectual disability, regression, cerebellar and cerebral atrophy, basal ganglia signal alterations, hypotonia, cerebellar signs, and increased blood lactate. In vitro studies showed that variants within the TARS2301-381 region had decreased binding to Rag GTPases, likely impairing mTORC1 activity. The zebrafish model recapitulated key features of the human phenotype and unraveled dysregulation of downstream targets of mTORC1 signaling. Functional testing of the variants confirmed the pathogenicity in a zebrafish model. CONCLUSION: We define the clinico-radiological spectrum of TARS2-related mitochondrial disease, unveil the likely involvement of the mTORC1 signaling pathway as a distinct molecular mechanism, and establish a TARS2 zebrafish model as an important tool to study variant pathogenicity.

Novel biallelic variants expand the phenotype of NAA20-related syndrome.

NAA20 is the catalytic subunit of the NatB complex, which is responsible for N-terminal acetylation of approximately 20% of the human proteome. Recently, pathogenic biallelic variants in NAA20 were associated with a novel neurodevelopmental disorder in five individuals with limited clinical information. We report two sisters harboring compound heterozygous variant (c.100C>T (p.Gln34Ter) and c.11T>C p.(Leu4Pro)) in the NAA20 gene, identified by exome sequencing. In vitro studies showed that the missense variant p.Leu4Pro resulted in a reduction of NAA20 catalytic activity due to weak coupling with the NatB auxiliary subunit. In addition, unpublished data of the previous families were reported, outlining the core phenotype of the NAA20-related disorder mostly characterized by cognitive impairment, microcephaly, ataxia, brain malformations, dysmorphism and variable occurrence of cardiac defect and epilepsy. Remarkably, our two patients featured epilepsy onset in adolescence suggesting this may be a part of syndrome evolution. Functional studies are needed to better understand the complexity of NAA20 variants pathogenesis as well as of other genes linked to N-terminal acetylation.

Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development.

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

De novo KCNA6 variants with attenuated KV 1.6 channel deactivation in patients with epilepsy.

OBJECTIVE: Mutations in the genes encoding neuronal ion channels are a common cause of Mendelian neurological diseases. We sought to identify novel de novo sequence variants in cases with early infantile epileptic phenotypes and neurodevelopmental anomalies. METHODS: Following clinical diagnosis, we performed whole exome sequencing of the index cases and their parents. Identified channel variants were expressed in Xenopus oocytes and their functional properties assessed using two-electrode voltage clamp. RESULTS: We identified novel de novo variants in KCNA6 in four unrelated individuals variably affected with neurodevelopmental disorders and seizures with onset in the first year of life. Three of the four identified mutations affect the pore-lining S6 α-helix of KV 1.6. A prominent finding of functional characterization in Xenopus oocytes was that the channel variants showed only minor effects on channel activation but slowed channel closure and shifted the voltage dependence of deactivation in a hyperpolarizing direction. Channels with a mutation affecting the S6 helix display dominant effects on channel deactivation when co-expressed with wild-type KV 1.6 or KV 1.1 subunits. SIGNIFICANCE: This is the first report of de novo nonsynonymous variants in KCNA6 associated with neurological or any clinical features. Channel variants showed a consistent effect on channel deactivation, slowing the rate of channel closure following normal activation. This specific gain-of-function feature is likely to underlie the neurological phenotype in our patients. Our data highlight KCNA6 as a novel channelopathy gene associated with early infantile epileptic phenotypes and neurodevelopmental anomalies.

mTORC1 functional assay reveals SZT2 loss-of-function variants and a founder in-frame deletion.

Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTORC1 signalling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions, but these variants are increasingly detected and reported by clinical genetic testing in individuals with epilepsy. To exemplify this latter point, here we describe a cohort of 12 individuals with biallelic SZT2 variants and phenotypic overlap with SZT2-related neurodevelopmental disorders. However, the majority of individuals carried one or more SZT2 variants of uncertain significance (VUS), highlighting the need for functional characterization to determine, which, if any, of these VUS were pathogenic. Thus, we developed a novel individualized platform to identify SZT2 loss-of-function variants in the context of mTORC1 signalling and reclassify VUS. Using this platform, we identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis revealed that this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Moreover, this approach allowed us to tentatively reclassify all of the VUS in our cohort of 12 individuals, identifying five individuals with biallelic pathogenic or likely pathogenic variants. Clinical features of these five individuals consisted of early-onset seizures (median 24 months), focal seizures, developmental delay and macrocephaly similar to previous reports. However, we also show a widening of the phenotypic spectrum, as none of the five individuals had corpus callosum abnormalities, in contrast to previous reports. Overall, we present a rapid assay to resolve VUS in SZT2, identify a founder variant in individuals of Ashkenazi Jewish ancestry, and demonstrate that corpus callosum abnormalities is not a hallmark feature of this condition. Our approach is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.

Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes.

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus.

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy and movement disorder. We evaluated a large cohort of patients (n = 25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor and ataxia. Later in the disease course, they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibres and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders.

De Novo Pathogenic Variants in N-cadherin Cause a Syndromic Neurodevelopmental Disorder with Corpus Collosum, Axon, Cardiac, Ocular, and Genital Defects.

Cadherins constitute a family of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The extracellular domain of cadherins consists of extracellular cadherin (EC) domains, separated by calcium binding sites. The EC interacts with other cadherin molecules in cis and in trans to mechanically hold apposing cell surfaces together. CDH2 encodes N-cadherin, whose essential roles in neural development include neuronal migration and axon pathfinding. However, CDH2 has not yet been linked to a Mendelian neurodevelopmental disorder. Here, we report de novo heterozygous pathogenic variants (seven missense, two frameshift) in CDH2 in nine individuals with a syndromic neurodevelopmental disorder characterized by global developmental delay and/or intellectual disability, variable axon pathfinding defects (corpus callosum agenesis or hypoplasia, mirror movements, Duane anomaly), and ocular, cardiac, and genital anomalies. All seven missense variants (c.1057G>A [p.Asp353Asn]; c.1789G>A [p.Asp597Asn]; c.1789G>T [p.Asp597Tyr]; c.1802A>C [p.Asn601Thr]; c.1839C>G [p.Cys613Trp]; c.1880A>G [p.Asp627Gly]; c.2027A>G [p.Tyr676Cys]) result in substitution of highly conserved residues, and six of seven cluster within EC domains 4 and 5. Four of the substitutions affect the calcium-binding site in the EC4-EC5 interdomain. We show that cells expressing these variants in the EC4-EC5 domains have a defect in cell-cell adhesion; this defect includes impaired binding in trans with N-cadherin-WT expressed on apposing cells. The two frameshift variants (c.2563_2564delCT [p.Leu855Valfs∗4]; c.2564_2567dupTGTT [p.Leu856Phefs∗5]) are predicted to lead to a truncated cytoplasmic domain. Our study demonstrates that de novo heterozygous variants in CDH2 impair the adhesive activity of N-cadherin, resulting in a multisystemic developmental disorder, that could be named ACOG syndrome (agenesis of corpus callosum, axon pathfinding, cardiac, ocular, and genital defects).

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

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.

Pathogenic variants in RNPC3 are associated with hypopituitarism and primary ovarian insufficiency.

PURPOSE: We aimed to investigate the molecular basis underlying a novel phenotype including hypopituitarism associated with primary ovarian insufficiency. METHODS: We used next-generation sequencing to identify variants in all pedigrees. Expression of Rnpc3/RNPC3 was analyzed by in situ hybridization on murine/human embryonic sections. CRISPR/Cas9 was used to generate mice carrying the p.Leu483Phe pathogenic variant in the conserved murine Rnpc3 RRM2 domain. RESULTS: We described 15 patients from 9 pedigrees with biallelic pathogenic variants in RNPC3, encoding a specific protein component of the minor spliceosome, which is associated with a hypopituitary phenotype, including severe growth hormone (GH) deficiency, hypoprolactinemia, variable thyrotropin (also known as thyroid-stimulating hormone) deficiency, and anterior pituitary hypoplasia. Primary ovarian insufficiency was diagnosed in 8 of 9 affected females, whereas males had normal gonadal function. In addition, 2 affected males displayed normal growth when off GH treatment despite severe biochemical GH deficiency. In both mouse and human embryos, Rnpc3/RNPC3 was expressed in the developing forebrain, including the hypothalamus and Rathke's pouch. Female Rnpc3 mutant mice displayed a reduction in pituitary GH content but with no reproductive impairment in young mice. Male mice exhibited no obvious phenotype. CONCLUSION: Our findings suggest novel insights into the role of RNPC3 in female-specific gonadal function and emphasize a critical role for the minor spliceosome in pituitary and ovarian development and function.

De novo missense variants in the E3 ubiquitin ligase adaptor KLHL20 cause a developmental disorder with intellectual disability, epilepsy, and autism spectrum disorder.

PURPOSE: KLHL20 is part of a CUL3-RING E3 ubiquitin ligase involved in protein ubiquitination. KLHL20 functions as the substrate adaptor that recognizes substrates and mediates the transfer of ubiquitin to the substrates. Although KLHL20 regulates neurite outgrowth and synaptic development in animal models, a role in human neurodevelopment has not yet been described. We report on a neurodevelopmental disorder caused by de novo missense variants in KLHL20. METHODS: Patients were ascertained by the investigators through Matchmaker Exchange. Phenotyping of patients with de novo missense variants in KLHL20 was performed. RESULTS: We studied 14 patients with de novo missense variants in KLHL20, delineating a genetic syndrome with patients having mild to severe intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, hyperactivity, and subtle dysmorphic facial features. We observed a recurrent de novo missense variant in 11 patients (NM_014458.4:c.1069G>A p.[Gly357Arg]). The recurrent missense and the 3 other missense variants all clustered in the Kelch-type ß-propeller domain of the KLHL20 protein, which shapes the substrate binding surface. CONCLUSION: Our findings implicate KLHL20 in a neurodevelopmental disorder characterized by intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, and hyperactivity.