SNUPN deficiency causes a recessive muscular dystrophy due to RNA mis-splicing and ECM dysregulation.

SNURPORTIN-1, encoded by SNUPN, plays a central role in the nuclear import of spliceosomal small nuclear ribonucleoproteins. However, its physiological function remains unexplored. In this study, we investigate 18 children from 15 unrelated families who present with atypical muscular dystrophy and neurological defects. Nine hypomorphic SNUPN biallelic variants, predominantly clustered in the last coding exon, are ascertained to segregate with the disease. We demonstrate that mutant SPN1 failed to oligomerize leading to cytoplasmic aggregation in patients' primary fibroblasts and CRISPR/Cas9-mediated mutant cell lines. Additionally, mutant nuclei exhibit defective spliceosomal maturation and breakdown of Cajal bodies. Transcriptome analyses reveal splicing and mRNA expression dysregulation, particularly in sarcolemmal components, causing disruption of cytoskeletal organization in mutant cells and patient muscle tissues. Our findings establish SNUPN deficiency as the genetic etiology of a previously unrecognized subtype of muscular dystrophy and provide robust evidence of the role of SPN1 for muscle homeostasis.

Molecular and Phenotypic Characterization of the RORB-Related Disorder.

BACKGROUND AND OBJECTIVES: Heterozygous variants in RAR-related orphan receptor B (RORB) have recently been associated with susceptibility to idiopathic generalized epilepsy. However, few reports have been published so far describing pathogenic variants of this gene in patients with epilepsy and intellectual disability (ID). In this study, we aimed to delineate the epilepsy phenotype associated with RORB pathogenic variants and to provide arguments in favor of the pathogenicity of variants. METHODS: Through an international collaboration, we analyzed seizure characteristics, EEG data, and genotypes of a cohort of patients with heterozygous variants in RORB. To gain insight into disease mechanisms, we performed ex vivo cortical electroporation in mouse embryos of 5 selected variants, 2 truncating and 3 missense, and evaluated on expression and quantified changes in axonal morphology. RESULTS: We identified 35 patients (17 male, median age 10 years, range 2.5-23 years) carrying 32 different heterozygous variants in RORB, including 28 single-nucleotide variants or small insertions/deletions (12 missense, 12 frameshift or nonsense, 2 splice-site variants, and 2 in-frame deletions), and 4 microdeletions; de novo in 18 patients and inherited in 10. Seizures were reported in 31/35 (89%) patients, with a median age at onset of 3 years (range 4 months-12 years). Absence seizures occurred in 25 patients with epilepsy (81%). Nineteen patients experienced a single seizure type: absences, myoclonic absences, or absences with eyelid myoclonia and focal seizures. Nine patients had absence seizures combined with other generalized seizure types. One patient had presented with absences associated with photosensitive occipital seizures. Three other patients had generalized tonic-clonic seizures without absences. ID of variable degree was observed in 85% of the patients. Expression studies in cultured neurons showed shorter axons for the 5 tested variants, both truncating and missense variants, supporting an impaired protein function. DISCUSSION: In most patients, the phenotype of the RORB-related disorder associates absence seizures with mild-to-moderate ID. In silico and in vitro evaluation of the variants in our cohort, including axonal morphogenetic experiments in cultured neurons, supports their pathogenicity, showing a hypomorphic effect.

Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.

Neurodevelopmental proteasomopathies represent a distinctive category of neurodevelopmental disorders (NDD) characterized by genetic variations within the 26S proteasome, a protein complex governing eukaryotic cellular protein homeostasis. In our comprehensive study, we identified 23 unique variants in PSMC5 , which encodes the AAA-ATPase proteasome subunit PSMC5/Rpt6, causing syndromic NDD in 38 unrelated individuals. Overexpression of PSMC5 variants altered human hippocampal neuron morphology, while PSMC5 knockdown led to impaired reversal learning in flies and loss of excitatory synapses in rat hippocampal neurons. PSMC5 loss-of-function resulted in abnormal protein aggregation, profoundly impacting innate immune signaling, mitophagy rates, and lipid metabolism in affected individuals. Importantly, targeting key components of the integrated stress response, such as PKR and GCN2 kinases, ameliorated immune dysregulations in cells from affected individuals. These findings significantly advance our understanding of the molecular mechanisms underlying neurodevelopmental proteasomopathies, provide links to research in neurodegenerative diseases, and open up potential therapeutic avenues.

Further evidence that the neurodevelopmental gene FBXW7 predisposes to Wilms tumor.

Somatic variants in the NOTCH pathway regulator FBXW7 are frequently seen in a variety of malignancies. Heterozygous loss-of-function germline variants in FBXW7 have recently been described as causative for a neurodevelopmental syndrome. Independently, FBXW7 was also considered as a susceptibility gene for Wilms tumor due to a few observations of heterozygous germline variants in patients with Wilms tumor. Whether the same FBXW7 variants are implicated in both, neurodevelopmental delay and Wilms tumor formation, remained unclear. By clinical testing, we now observed a patient with neurodevelopmental delay due to a de novo constitutional mosaic FBXW7 splice site pathogenic variant who developed Wilms tumor. In the tumor, we identified a second hit frameshift variant in FBXW7. Immunohistochemical staining was consistent with mosaic loss of FBXW7 protein expression in the tumor. Our data support the role of constitutional FBXW7 pathogenic variants in both, neurodevelopmental disorder and the etiology of Wilms tumor. Therefore, Wilms tumor screening should be considered in individuals with constitutional or germline pathogenic variants in FBXW7 and associated neurodevelopmental syndrome.

Biallelic MED27 variants lead to variable ponto-cerebello-lental degeneration with movement disorders.

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants. Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 ± 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed. MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed. This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'.

A very mild phenotype in six individuals of a three-generation family with the novel HRAS variant c.176C > G p.(Ala59Gly): Emergence of a new HRAS-related RASopathy distinct from Costello syndrome.

Costello syndrome is a clinically recognizable, severe neurodevelopmental disorder caused by heterozygous activating variants in HRAS. The vast majority of affected patients share recurring variants affecting HRAS codons 12 and 13 and a relatively uniform phenotype. Here, we report the unique and attenuated phenotype of six individuals of an extended family affected by the HRAS variant c.176C>T p.(Ala59Gly), which, to our knowledge, has never been reported as a germline variant in patients so far. HRAS Alanine 59 has been previously functionally investigated as an oncogenic hotspot and the p.Ala59Gly substitution was shown to impair intrinsic GTP hydrolysis. All six individuals we report share a phenotype of ectodermal anomalies and mild features suggestive of a RASopathy, reminiscent of patients with Noonan syndrome-like disorder with loose anagen hair. All six are of normal intelligence, none have a history of failure to thrive or malignancy, and they have no known cardiac or neurologic pathologies. Our report adds to the previous reports of patients with rare variants affecting amino acids located in the SWITCH II/G3 region of HRAS and suggests a consistent, attenuated phenotype distinct from classical Costello syndrome. We propose the definition of a new distinct HRAS-related RASopathy for patients carrying HRAS variants affecting codons 58, 59, 60.

The genetic landscape and clinical implication of pediatric Moyamoya angiopathy in an international cohort.

Pediatric Moyamoya Angiopathy (MMA) is a progressive intracranial occlusive arteriopathy that represents a leading cause of transient ischemic attacks and strokes in childhood. Despite this, up to now no large, exclusively pediatric MMA cohort has been subjected to systematic genetic investigation. In this study, we performed molecular karyotyping, exome sequencing and automated structural assessment of missense variants on a series of 88 pediatric MMA patients and correlated genetic, angiographic and clinical (stroke burden) findings. The two largest subgroups in our cohort consisted of RNF213 and neurofibromatosis type 1 (NF1) patients. While deleterious RNF213 variants were associated with a severe MMA clinical course with early symptom onset, frequent posterior cerebral artery involvement and higher stroke rates in multiple territories, NF1 patients had a similar infarct burden compared to non-NF1 individuals and were often diagnosed incidentally during routine MRIs. Additionally, we found that MMA-associated RNF213 variants have lower predicted functional impact compared to those associated with aortic disease. We also raise the question of MMA as a feature of recurrent as well as rare chromosomal imbalances and further support the possible association of MMA with STAT3 deficiency. In conclusion, we provide a comprehensive characterization at the genetic and clinical level of a large exclusively pediatric MMA population. Due to the clinical differences found across genetic subgroups, we propose genetic testing for risk stratification as part of the routine assessment of pediatric MMA patients.

The current benefit of genome sequencing compared to exome sequencing in patients with developmental or epileptic encephalopathies.

BACKGROUND: As the technology of next generation sequencing rapidly develops and costs are constantly reduced, the clinical availability of whole genome sequencing (WGS) increases. Thereby, it remains unclear what exact advantage WGS offers in comparison to whole exome sequencing (WES) for the diagnosis of genetic diseases using current technologies. METHODS: Trio-WGS was conducted for 20 patients with developmental or epileptic encephalopathies who remained undiagnosed after WES and chromosomal microarray analysis. RESULTS: A diagnosis was reached for four patients (20%). However, retrospectively all pathogenic variants could have been detected in a WES analysis conducted with today's methods and knowledge. CONCLUSION: The additional diagnostic yield of WGS versus WES is currently largely explained by new scientific insights and the general technological progress. Nevertheless, it is noteworthy that whole genome sequencing has greater potential for the analysis of small copy number and copy number neutral variants not seen with WES as well as variants in noncoding regions, especially as potentially more knowledge of the function of noncoding regions arises. We, therefore, conclude that even though today the added value of WGS versus WES seems to be limited, it may increase substantially in the future.

A clustering of heterozygous missense variants in the crucial chromatin modifier WDR5 defines a new neurodevelopmental disorder.

WDR5 is a broadly studied, highly conserved key protein involved in a wide array of biological functions. Among these functions, WDR5 is a part of several protein complexes that affect gene regulation via post-translational modification of histones. We collected data from 11 unrelated individuals with six different rare de novo germline missense variants in WDR5; one identical variant was found in five individuals and another variant in two individuals. All individuals had neurodevelopmental disorders including speech/language delays (n = 11), intellectual disability (n = 9), epilepsy (n = 7), and autism spectrum disorder (n = 4). Additional phenotypic features included abnormal growth parameters (n = 7), heart anomalies (n = 2), and hearing loss (n = 2). Three-dimensional protein structures indicate that all the residues affected by these variants are located at the surface of one side of the WDR5 protein. It is predicted that five out of the six amino acid substitutions disrupt interactions of WDR5 with RbBP5 and/or KMT2A/C, as part of the COMPASS (complex proteins associated with Set1) family complexes. Our experimental approaches in Drosophila melanogaster and human cell lines show normal protein expression, localization, and protein-protein interactions for all tested variants. These results, together with the clustering of variants in a specific region of WDR5 and the absence of truncating variants so far, suggest that dominant-negative or gain-of-function mechanisms might be at play. All in all, we define a neurodevelopmental disorder associated with missense variants in WDR5 and a broad range of features. This finding highlights the important role of genes encoding COMPASS family proteins in neurodevelopmental disorders.

Biallelic PRMT7 pathogenic variants are associated with a recognizable syndromic neurodevelopmental disorder with short stature, obesity, and craniofacial and digital abnormalities.

PURPOSE: Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyzes the methylation of arginine residues on several protein substrates. Biallelic pathogenic PRMT7 variants have previously been associated with a syndromic neurodevelopmental disorder characterized by short stature, brachydactyly, intellectual developmental disability, and seizures. To our knowledge, no comprehensive study describes the detailed clinical characteristics of this syndrome. Thus, we aim to delineate the phenotypic spectrum of PRMT7-related disorder. METHODS: We assembled a cohort of 51 affected individuals from 39 different families, gathering clinical information from 36 newly described affected individuals and reviewing data of 15 individuals from the literature. RESULTS: The main clinical characteristics of the PRMT7-related syndrome are short stature, mild to severe developmental delay/intellectual disability, hypotonia, brachydactyly, and distinct facial morphology, including bifrontal narrowing, prominent supraorbital ridges, sparse eyebrows, short nose with full/broad nasal tip, thin upper lip, full and everted lower lip, and a prominent or squared-off jaw. Additional variable findings include seizures, obesity, nonspecific magnetic resonance imaging abnormalities, eye abnormalities (i.e., strabismus or nystagmus), and hearing loss. CONCLUSION: This study further delineates and expands the molecular, phenotypic spectrum and natural history of PRMT7-related syndrome characterized by a neurodevelopmental disorder with skeletal, growth, and endocrine abnormalities.

Microdeletions at 19p13.11p12 in five individuals with neurodevelopmental delay.

Only few copy number variants at chromosome 19p13.11 have been reported, thus associated clinical information is scarce. Proximal to these copy number losses, we now identified deletions in five unrelated individuals with neurodevelopmental disorders. They presented with psychomotor delay as well as behavioral and sleeping disorders, while complex cardiovascular, skeletal, and various other malformations were more variable. Dysmorphic features were rather unspecific and not considered as a recognizable gestalt. Neither of the analyzed parents carried their offsprings' deletions, indicating de novo occurrence. The deletion sizes ranged between 0.7 and 5.2 Mb, were located between 18 and 24 megabases from the telomere, and contained a variable number of protein-coding genes (n = 25-68). Although not all microdeletions shared a common region, the smallest common overlap of some of the deletions provided interesting insights in the chromosomal region 19p13.11p12. Diligent literature review using OMIM and Pubmed did not identify a satisfying candidate gene for neurodevelopmental disorders. In the literature, a de novo in-frame deletion in MAU2 was considered pathogenic in an individual with Cornelia de Lange syndrome. Therefore, the clinical differential diagnosis of this latter syndrome in one individual and the encompassment of MAU2 in three individuals' deletions suggest clinical and genetic overlap with this specific syndrome. Three of the four here reported individuals with deletion encompassing GDF1 had different congenital heart defects, suggesting that this gene's haploinsufficiency might contribute to the cardiovascular phenotype, however, with reduced penetrance. Our findings indicate an association of microdeletions at 19p13.11/ 19p13.11p12 with neurodevelopmental disorders, variable symptoms, and malformations, and delineate the phenotypic spectrum of deletions within this genomic region.

Case report: ETS1 gene deletion associated with a low number of recent thymic emigrants in three patients with Jacobsen syndrome.

Jacobsen syndrome is a rare genetic disorder associated with a terminal deletion in chromosome 11. The clinical presentation is variable. Although immunodeficiency has been described in patients with Jacobsen syndrome, a clear genotype-phenotype correlation has not yet been established. Here, we report on the immunologic phenotypes of four patients with Jacobsen syndrome. All four patients showed one or more atypical immunologic features. One patient suffered from recurrent viral infections, two patients had experienced a severe bacterial infection and one had received antibiotic prophylaxis since early childhood. One patient had experienced severe, transient immune dysregulation. Hypogammaglobulinemia and low B cell counts were found in two patients, while the number of recent thymic emigrants (CD31+CD45RA+ CD4 cells) was abnormally low in three. When considering the six immune-related genes located within the affected part of chromosome 11 (ETS1, TIRAP, FLI1, NFRKB, THYN1, and SNX19), only the ETS1 gene was found be deleted in the three patients with low numbers of recent thymic emigrants and non-switched memory B cells. Our findings support the hypothesis whereby Jacobsen syndrome is associated with a combined immunodeficiency with variable presentation. Further investigations of potential genotype-phenotype correlations are warranted and might help to personalize patient management in individuals lacking immune-related genes. In addition, we recommend immunologic follow-up for all patients with Jacobsen syndrome, as immune abnormalities may develop over time.

Bi-allelic variants in DOHH, catalyzing the last step of hypusine biosynthesis, are associated with a neurodevelopmental disorder.

Deoxyhypusine hydroxylase (DOHH) is the enzyme catalyzing the second step in the post-translational synthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] in the eukaryotic initiation factor 5A (eIF5A). Hypusine is formed exclusively in eIF5A by two sequential enzymatic steps catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusinated eIF5A is essential for translation and cell proliferation in eukaryotes, and all three genes encoding eIF5A, DHPS, and DOHH are highly conserved throughout eukaryotes. Pathogenic variants affecting either DHPS or EIF5A have been previously associated with neurodevelopmental disorders. Using trio exome sequencing, we identified rare bi-allelic pathogenic missense and truncating DOHH variants segregating with disease in five affected individuals from four unrelated families. The DOHH variants are associated with a neurodevelopmental phenotype that is similar to phenotypes caused by DHPS or EIF5A variants and includes global developmental delay, intellectual disability, facial dysmorphism, and microcephaly. A two-dimensional gel analyses revealed the accumulation of deoxyhypusine-containing eIF5A [eIF5A(Dhp)] and a reduction in the hypusinated eIF5A in fibroblasts derived from affected individuals, providing biochemical evidence for deficiency of DOHH activity in cells carrying the bi-allelic DOHH variants. Our data suggest that rare bi-allelic variants in DOHH result in reduced enzyme activity, limit the hypusination of eIF5A, and thereby lead to a neurodevelopmental disorder.

Assessing clinical utility of preconception expanded carrier screening regarding residual risk for neurodevelopmental disorders.

The magnitude of clinical utility of preconception expanded carrier screening (ECS) concerning its potential to reduce the risk of affected offspring is unknown. Since neurodevelopmental disorders (NDDs) in their offspring is a major concern of parents-to-be, we addressed the question of residual risk by assessing the risk-reduction potential for NDDs in a retrospective study investigating ECS with different criteria for gene selection and definition of pathogenicity. We used exome sequencing data from 700 parents of children with NDDs and blindly screened for carrier-alleles in up to 3046 recessive/X-linked genes. Depending on variant pathogenicity thresholds and gene content, NDD-risk-reduction potential was up to 43.5% in consanguineous, and 5.1% in nonconsanguineous couples. The risk-reduction-potential was compromised by underestimation of pathogenicity of missense variants (false-negative-rate 4.6%), inherited copy-number variants and compound heterozygosity of one inherited and one de novo variant (0.9% each). Adherence to the ACMG recommendations of restricting ECS to high-frequency genes in nonconsanguineous couples would more than halve the detectable inherited NDD-risk. Thus, for optimized clinical utility of ECS, screening in recessive/X-linked genes regardless of their frequency (ACMG Tier-4) and sensible pathogenicity thresholds should be considered for all couples seeking ECS.

Further characterization of Borjeson-Forssman-Lehmann syndrome in females due to de novo variants in PHF6.

While inherited hemizygous variants in PHF6 cause X-linked recessive Borjeson-Forssman-Lehmann syndrome (BFLS) in males, de novo heterozygous variants in females are associated with an overlapping but distinct phenotype, including moderate to severe intellectual disability, characteristic facial dysmorphism, dental, finger and toe anomalies, and linear skin pigmentation. By personal communication with colleagues, we assembled 11 additional females with BFLS due to variants in PHF6. We confirm the distinct phenotype to include variable intellectual disability, recognizable facial dysmorphism and other anomalies. We observed skewed X-inactivation in blood and streaky skin pigmentation compatible with functional mosaicism. Variants occurred de novo in 10 individuals, of whom one was only mildly affected and transmitted it to her more severely affected daughter. The mutational spectrum comprises a two-exon deletion, five truncating, one splice-site and three missense variants, the latter all located in the PHD2 domain and predicted to severely destabilize the domain structure. This observation supports the hypothesis of more severe variants in females contributing to gender-specific phenotypes in addition to or in combination with effects of X-inactivation and functional mosaicism. Therefore, our findings further delineate the clinical and mutational spectrum of female BFLS and provide further insights into possible genotype-phenotype correlations between females and males.

The MAP3K7 gene: Further delineation of clinical characteristics and genotype/phenotype correlations.

Mitogen-activated protein 3 kinase 7 (MAP3K7) encodes the ubiquitously expressed transforming growth factor ß-activated kinase 1, which plays a crucial role in many cellular processes. Mutationsin the MAP3K7 gene have been linked to two distinct disorders: frontometaphyseal dysplasia type 2 (FMD2) and cardiospondylocarpofacial syndrome (CSCF). The fact that different mutations can induce two distinct phenotypes suggests a phenotype/genotype correlation, but no side-by-side comparison has been done thus far to confirm this. Here, we significantly expand the cohort and the description of clinical phenotypes for patients with CSCF and FMD2 who carry mutations in MAP3K7. Our findings support that in contrast to FMD2-causing mutations, CSCF-causing mutations in MAP3K7 have a loss-of-function effect. Additionally, patients with pathogenic mutations in MAP3K7 are at risk for (severe) cardiac disease, have symptoms associated with connective tissue disease, and we show overlap in clinical phenotypes of CSCF with Noonan syndrome (NS). Together, we confirm a molecular fingerprint of FMD2- versus CSCF-causing MAP3K7 mutations and conclude that mutations in MAP3K7 should be considered in the differential diagnosis of patients with syndromic congenital cardiac defects and/or cardiomyopathy, syndromic connective tissue disorders, and in the differential diagnosis of NS.

Human COQ4 deficiency: delineating the clinical, metabolic and neuroimaging phenotypes.

BACKGROUND: Human coenzyme Q4 (COQ4) is essential for coenzyme Q10 (CoQ10) biosynthesis. Pathogenic variants in COQ4 cause childhood-onset neurodegeneration. We aimed to delineate the clinical spectrum and the cellular consequences of COQ4 deficiency. METHODS: Clinical course and neuroradiological findings in a large cohort of paediatric patients with COQ4 deficiency were analysed. Functional studies in patient-derived cell lines were performed. RESULTS: We characterised 44 individuals from 36 families with COQ4 deficiency (16 newly described). A total of 23 different variants were identified, including four novel variants in COQ4. Correlation analyses of clinical and neuroimaging findings revealed three disease patterns: type 1: early-onset phenotype with neonatal brain anomalies and epileptic encephalopathy; type 2: intermediate phenotype with distinct stroke-like lesions; and type 3: moderate phenotype with non-specific brain pathology and a stable disease course. The functional relevance of COQ4 variants was supported by in vitro studies using patient-derived fibroblast lines. Experiments revealed significantly decreased COQ4 protein levels, reduced levels of cellular CoQ10 and elevated levels of the metabolic intermediate 6-demethoxyubiquinone. CONCLUSION: Our study describes the heterogeneous clinical presentation of COQ4 deficiency and identifies phenotypic subtypes. Cell-based studies support the pathogenic characteristics of COQ4 variants. Due to the insufficient clinical response to oral CoQ10 supplementation, alternative treatment strategies are warranted.