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Purpose To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons. Methods We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity. Results We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation. Conclusions Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS.

Protein translation is an essential cellular process and dysfunctional protein translation causes various neurodevelopmental disorders. The eukaryotic translation elongation factor 1A (eEF1A) delivers aminoacyl-tRNA to the ribosome, while the eEF1B complex acts as a guanine exchange factor (GEF) of GTP for GDP indirectly catalyzing the release of eEF1A from the ribosome. The gene EEF1D encodes the eEF1Bδ subunit of the eEF1B complex. EEF1D is alternatively spliced giving rise to one long and three short isoforms. Two different homozygous, truncating variants in EEF1D had been associated with severe intellectual disability and microcephaly in two families. The published variants only affect the long isoform of EEF1D that acts as a transcription factor of heat shock element proteins. By exome sequencing, we identified two different homozygous variants in EEF1D in two families with severe developmental delay, severe microcephaly, spasticity, and failure to thrive with optic atrophy, poor feeding, and recurrent aspiration pneumonias. The EEF1D variants of this study are localized in the C-terminal GEF domain suggesting that a disturbed protein translation machinery might contribute to the neurodevelopmental phenotype. Pathogenic variants localized in both the alternatively spliced domain or in the GEF domain of EEF1D cause a severe neurodevelopmental disorder with microcephaly and spasticity. This article is protected by copyright. All rights reserved.

The neuronal SNARE complex drives synaptic vesicle exocytosis. Therefore, one of its core proteins syntaxin 1A (STX1A) has long been suspected to play a role in neurodevelopmental disorders. We assembled eight individuals harboring ultra rare variants in STX1A who present with a spectrum of intellectual disability, autism and epilepsy. Causative variants comprise a homozygous splice variant, three de novo missense variants and two inframe deletions of a single amino acid. We observed a phenotype mainly driven by epilepsy in the individuals with missense variants in contrast to intellectual disability and autistic behavior in individuals with single amino acid deletions and the splicing variant. In silico modeling of missense variants and single amino acid deletions show different impaired protein-protein interactions. We hypothesize the two phenotypic courses of affected individuals to be dependent on two different pathogenic mechanisms: (1) a weakened inhibitory STX1A-STXBP1 interaction due to missense variants results in an STX1A-related developmental epileptic encephalopathy and (2) a hampered SNARE complex formation due to inframe deletions causes an STX1A-related intellectual disability and autism phenotype. Our description of a STX1A-related neurodevelopmental disorder with or without epilepsy thus expands the group of rare diseases called SNAREopathies.

Purpose: The study aimed to investigate the role of PABPC1 in developmental delay (DD). Methods: Children were examined by geneticists and pediatricians. Variants were identified using exome sequencing and standard downstream bioinformatics pipelines. We performed in silico molecular modeling and coimmunoprecipitation to test if the variants affect the interaction between PABPC1 and PAIP2. We performed in utero electroporation of mouse embryo brains to enlighten the function of PABPC1. Results: We describe 4 probands with an overlapping phenotype of DD, expressive speech delay, and autistic features and heterozygous de novo variants that cluster in the PABP domain of PABPC1. Further symptoms were seizures and behavioral disorders. Molecular modeling predicted that the variants are pathogenic and would lead to decreased binding affinity to messenger RNA metabolism-related proteins, such as PAIP2. Coimmunoprecipitation confirmed this because it showed a significant weakening of the interaction between mutant PABPC1 and PAIP2. Electroporation of mouse embryo brains showed that Pabpc1 knockdown decreases the proliferation of neural progenitor cells. Wild-type Pabpc1 could rescue this disturbance, whereas 3 of the 4 variants did not. Conclusion: Pathogenic variants in the PABP domain lead to DD, possibly because of interference with the translation initiation and subsequently an impaired neurogenesis in cortical development.

Most individuals with rare diseases initially consult their primary care physician. For a subset of rare diseases, efficient diagnostic pathways are available. However, ultra-rare diseases often require both expert clinical knowledge and comprehensive genetic diagnostics, which poses structural challenges for public healthcare systems. To address these challenges within Germany, a novel structured diagnostic concept, based on multidisciplinary expertise at established university hospital centers for rare diseases (CRDs), was evaluated in the three year prospective study TRANSLATE NAMSE. A key goal of TRANSLATE NAMSE was to assess the clinical value of exome sequencing (ES) in the ultra-rare disease population. The aims of the present study were to perform a systematic investigation of the phenotypic and molecular genetic data of TRANSLATE NAMSE patients who had undergone ES in order to determine the yield of both ultra-rare diagnoses and novel gene-disease associations; and determine whether the complementary use of machine learning and artificial intelligence (AI) tools improved diagnostic effectiveness and efficiency.ES was performed for 1,577 patients (268 adult and 1,309 pediatric). Molecular genetic diagnoses were established in 499 patients (74 adult and 425 pediatric). A total of 370 distinct molecular genetic causes were established. The majority of these concerned known disorders, most of which were ultra-rare. During the diagnostic process, 34 novel and 23 candidate genotype-phenotype associations were delineated, mainly in individuals with neurodevelopmental disorders.To determine the likelihood that ES will lead to a molecular diagnosis in a given patient, based on the respective clinical features only, we developed a statistical framework called YieldPred. The genetic data of a subcohort of 224 individuals that also gave consent to the computer-assisted analysis of their facial images were processed with the AI tool Prioritization of Exome Data by Image Analysis (PEDIA) and showed superior performance in variant prioritization.The present analyses demonstrated that the novel structured diagnostic concept facilitated the identification of ultra-rare genetic disorders and novel gene-disease associations on a national level and that the machine learning and AI tools improved diagnostic effectiveness and efficiency for ultra-rare genetic disorders.

We report two series of individuals with DDX3X variations, one (48 individuals) from physicians and one (44 individuals) from caregivers. These two series include several symptoms in common, with fairly similar distribution, which suggests that caregivers’ data are close to physicians’ data. For example, both series identified early childhood symptoms that were not previously described: feeding difficulties, mean walking age and age at first words. Each of the two datasets provide complementary knowledge. We confirmed that symptoms are similar to those in the literature and provide more details on feeding difficulties. Caregivers considered that the symptom attention-deficit/hyperactivity disorder was most worrisome. Both series also reported sleep disturbance. Recently, anxiety has been reported in individuals with DDX3X variants. We strongly suggest that attention-deficit/hyperactivity disorder, anxiety and sleep disorders need to be treated. In addition, we demonstrate preliminary evidence of a mild genome-wide DNA methylation profile in patients carrying mutations in DDX3X.

PURPOSECoffin-Siris and Nicolaides-Baraitser syndromes, are recognisable neurodevelopmental disorders caused by germline variants in BAF complex subunits. TheSMARCC2BAFopathy was recently reported. Herein, we present clinical and molecular data on a large cohort.METHODSClinical symptoms for 41 novel and 24 previously published cases were analyzed using the Human Phenotype Ontology. For genotype-phenotype correlation, molecular data were standardized and grouped into non-truncating and likely gene-disrupting variants (LGD). Missense variant protein expression and BAF subunit interactions were examined using 3D protein modeling, co-immunoprecipitation, and proximity-ligation assays.RESULTSNeurodevelopmental delay with intellectual disability, muscular hypotonia and behavioral disorders were the major manifestations. Clinical hallmarks of BAFopathies were rare. Clinical presentation differed significantly, with LGD variants being predominantly inherited and associated with mildly reduced or normal cognitive development, while non-truncating variants were mostlyde novoand presented with severe developmental delay. These distinct manifestations and non-truncating variant clustering in functional domains suggest different pathomechanisms.In vitrotesting showed decreased protein expression for N-terminal missense variants similar to LGD.CONCLUSIONThis study improvedSMARCC2variant classification and identified discernibleSMARCC2-associated phenotypes for LGD and non-truncating variants, which were distinct from other BAFopathies. The pathomechanism of most non-truncating variants has yet to be investigated.

The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.

Eukaryotic initiation factor-4A2 (EIF4A2) is an ATP-dependent RNA helicase and a member of the DEAD-box protein family that recognizes the 5' cap structure of mRNAs, allows mRNA to bind to the ribosome, and plays an important role in microRNA-regulated gene repression. Here, we report on 15 individuals from 14 families presenting with global developmental delay, intellectual disability, hypotonia, epilepsy, and structural brain anomalies, all of whom have extremely rare de novo mono-allelic or inherited bi-allelic variants in EIF4A2. Neurodegeneration was predominantly reported in individuals with bi-allelic variants. Molecular modeling predicts these variants would perturb structural interactions in key protein domains. To determine the pathogenicity of the EIF4A2 variants in vivo, we examined the mono-allelic variants in Drosophila melanogaster (fruit fly) and identified variant-specific behavioral and developmental defects. The fruit fly homolog of EIF4A2 is eIF4A, a negative regulator of decapentaplegic (dpp) signaling that regulates embryo patterning, eye and wing morphogenesis, and stem cell identity determination. Our loss-of-function (LOF) rescue assay demonstrated a pupal lethality phenotype induced by loss of eIF4A, which was fully rescued with human EIF4A2 wild-type (WT) cDNA expression. In comparison, the EIF4A2 variant cDNAs failed or incompletely rescued the lethality. Overall, our findings reveal that EIF4A2 variants cause a genetic neurodevelopmental syndrome with both LOF and gain of function as underlying mechanisms.

Biallelic PNKP variants cause heterogeneous disorders ranging from neurodevelopmental disorder with microcephaly/seizures to adult-onset Charcot–Marie–Tooth disease. To date, only postnatal descriptions exist. We present the first prenatal diagnosis of PNKP-related primary microcephaly. Pathological examination of a male fetus in the 18th gestational week revealed micrencephaly with extracerebral malformations and thus presumed syndromic microcephaly. A recessive disorder was suspected because of previous pregnancy termination for similar abnormalities. Prenatal trio-exome sequencing identified compound heterozygosity for the PNKP variants c.498G>A, p.[(=),0?] and c.302C>T, p.(Pro101Leu). Segregation confirmed both variants in the sister fetus. Through RNA analyses, we characterized exon 4 skipping affecting the PNKP forkhead-associated (FHA) and phosphatase domains (p.Leu67_Lys166del) as the predominant effect of the paternal c.498G>A variant. We retrospectively investigated two unrelated individuals diagnosed with biallelic PNKP-variants to compare prenatal/postnatal phenotypes. Both carry the splice donor variant c.1029+2T>C intrans with a variant in the FHA domain (c.311T>C, p.(Leu104Pro); c.151G>C, p.(Val51Leu)). RNA-seq showed complex splicing for c.1029+2T>C and c.151G>C. Structural modeling revealed significant clustering of missense variants in the FHA domain with variants generating structural damage. Our clinical description extends the PNKP-continuum to the prenatal stage. Investigating possible PNKP-variant effects using RNA and structural modeling, we highlight the mutational complexity and exemplify a PNKP-variant characterization framework.

ZUSAMMENFASSUNGHoloprosenzephalie (HPE) ist ein komplexes Krankheitsbild mit variabler genetischer und klinischer Ausprägung. Es existieren verschiedene Formen der HPE mit Unterschieden in der Schwere des Verlaufes. Die schwere Form der HPE stellen die lobare und alobare HPE dar, die mit Zyklopie (Vereinigung von beiden Augenanlagen in einer einzigen Orbita) einhergehen, wohingegen Minor-Formen/HPE-Spektrumerkrankungen eine Mikrozephalie und/oder Hypotelorismus oder eine Lippen-Kiefer-Gaumenspalte aufweisen. Die genetischen Mechanismen sind noch nicht abschließend geklärt. Es wurden autosomal-dominante, rezessive und digenetische Vererbungsmuster beschrieben. Wir ergänzen das Spektrum der HPE-Erkrankungen um eine Patientin mit compound heterozygoter DISP1-Mutation, die einen Panhypopituitarismus in Kombination mit Entwicklungverzögerung, Lippen-Kiefer-Gaumenspalte, persistierendem Ductus Arteriosus Botalli und Visusminderung aufweist.

Purpose Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants. Methods We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality. Results Patients with variants in GDF11 presented with craniofacial (5/6) , vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6) and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients’ variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) alleles whereas the missense variants in our cohort are partial LOF variants. Conclusion GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.

Heterozygous, pathogenic CUX1 variants are associated with global developmental delay or intellectual disability. This study delineates the clinical presentation in an extended cohort and investigates the molecular mechanism underlying the disorder in a Cux1+/− mouse model. Through international collaboration, we assembled the phenotypic and molecular information for 34 individuals (23 unpublished cases). A Cux1+/− mouse model was used to analyze CUX1 expression in the brain and evaluate susceptibility to epilepsy. We describe 34 patients with 26 different null and four missense variants. The leading symptoms were mild to moderate delayed speech and motor development, and intellectual disability. In Cux1+/− mice, we found delayed growth, histologically normal brains, and increased susceptibility to seizures. In Cux1+/− brains, the expression of Cux1 transcripts was half of WT animals. Expression of CUX1 proteins was also reduced, although in early postnatal animals significantly more than in adults. In summary, disease-causing CUX1 variants result in a non-syndromic phenotype of developmental delay and intellectual disability. In some patients, this phenotype ameliorates with age, resulting in a clinical catch-up and normal IQ in adulthood. Furthermore, the balance of CUX1 isoform expression in the brain during development appears to be important for this favorable clinical course.

Objective: Rare inherited missense variants in SLC32A1, the gene that encodes the vesicular gamma-aminobutyric acid (GABA) transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment.Methods: Using exome sequencing, we identified four individuals with a developmental and epileptic encephalopathy and de novo missense variants in SLC32A1. To assess causality, we performed functional evaluation of the identified variants in a murine neuronal cell culture model.Results: The main phenotype comprises moderate-to-severe intellectual disability, infantile-onset epilepsy within the first 18 months of life, and a choreiform, dystonic, or dyskinetic movement disorder. In silico modeling and functional analyses reveal that three of these variants, which are located in helices that line the putative GABA transport pathway, result in reduced quantal size, consistent with impaired filling of synaptic vesicles with GABA. The fourth variant, located in the vesicular gamma-aminobutyric acid N-terminus, does not affect quantal size, but increases presynaptic release probability, leading to more severe synaptic depression during high-frequency stimulation. Thus, variants in vesicular gamma-aminobutyric acid can impair GABAergic neurotransmission through at least two mechanisms, by affecting synaptic vesicle filling and by altering synaptic short-term plasticity.Interpretation: This work establishes de novo missense variants in SLC32A1 as a novel cause of a developmental and epileptic encephalopathy. SUMMARY FOR SOCIAL MEDIA IF PUBLISHED: @platzer_k @lemke_johannes @RamiJamra @Nirgalito @GeneDx The SLC family 32 Member 1 (SLC32A1) is the only protein identified to date, that loads gamma-aminobutyric acid (GABA) and glycine into synaptic vesicles, and is therefore also known as the vesicular GABA transporter (VGAT) or vesicular inhibitory amino acid transporter (VIAAT). Rare inherited missense variants in SLC32A1, the gene that encodes VGAT/vesicular inhibitory amino acid transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. We report on four individuals with de novo missense variants in SLC32A1 and a developmental and epileptic encephalopathy with infantile onset epilepsy. We establish causality of the variants via in silico modeling and their functional evaluation in a murine neuronal cell culture model. SLC32A1 variants represent a novel genetic etiology in neurodevelopmental disorders with epilepsy and a new GABA-related disease mechanism.

Routine diagnostics is biased towards genes and variants with satisfactory evidence, but rare disorders with only little confirmation of their pathogenicity might be missed. Many of these genes can, however, be considered relevant, although they may have less evidence because they lack OMIM entries or comprise only a small number of publicly available variants from one or a few studies. Here, we present 89 individuals harbouring variants in 77 genes for which only a small amount of public evidence on their clinical significance is available but which we still found to be relevant enough to be reported in routine diagnostics. For 21 genes, we present case reports that confirm the lack or provisionality of OMIM associations (ATP6V0A1, CNTN2, GABRD, NCKAP1, RHEB, TCF7L2), broaden the phenotypic spectrum (CC2D1A, KCTD17, YAP1) or substantially strengthen the confirmation of genes with limited evidence in the medical literature (ADARB1, AP2M1, BCKDK, BCORL1, CARS2, FBXO38, GABRB1, KAT8, PRKD1, RAB11B, RUSC2, ZNF142). Routine diagnostics can provide valuable information on disease associations and support for genes without requiring tremendous research efforts. Thus, our results validate and delineate gene–disorder associations with the aim of motivating clinicians and scientists in diagnostic departments to provide additional evidence via publicly available databases or by publishing short case reports.

Iterative re-analysis of NGS results is not well investigated for published research cohorts of rare diseases.We revisited a cohort of 152 consanguineous families with developmental disorders (NDD) reported five years ago. We re-evaluated all reported variants according to diagnostic classification guidelines or our candidate gene scoring system (AutoCaSc) and systematically scored the validity of gene-disease associations. Sequencing data was re-processed using an up-to-date pipeline for case-level re-analysis.In 30/152 (20%) families, we identified a clinically relevant change. Thirteen previously reported (likely) pathogenic variants were re-classified as VUS/benign. In three cases, the gene-disease association (TSEN15, NAPB, and FAR1) validity was judged as limited. We identified 12 new disease causing variants. Two previously reported variants were missed by our updated pipeline due to alignment or reference issues.Our results support the need to re-evaluate screening studies, not only the negative cases but including supposedly solved ones. This also applies in a diagnostic setting. We highlight that the complexity of computational re-analysis for old data should be weighed against the decreasing re-testing costs. Since extensive re-analysis per case is beyond the resources of most institutions, we recommend a screening procedure that would quickly identify the majority (83%) of new variants.

Purpose Genetic diagnostics of neurodevelopmental disorders with epilepsy (NDDE) are predominantly applied in children, thus limited information is available regarding adults or elderly. Methods We investigated 150 adult/elderly individuals with NDDE by conventional karyotyping, FMR1 testing, chromosomal microarray, panel sequencing, and for unresolved cases, also by exome sequencing (nsingle = 71, ntrios = 24). Results We identified (likely) pathogenic variants in 71 cases (47.3%) comprising fragile X syndrome (n = 1), disease-causing copy number (n = 23), and single-nucleotide variants (n = 49). Seven individuals displayed multiple independent genetic diagnoses. The diagnostic yield correlated with the severity of intellectual disability. Individuals with anecdotal evidence of exogenic early-life events (e.g., nuchal cord, complications at delivery) with alleged/unproven association to the disorder had a particularly high yield of 58.3%. Screening for disease-specific comorbidities was indicated in 45.1% and direct treatment consequences arose in 11.8% of diagnosed individuals. Conclusion Panel/exome sequencing displayed the highest yield and should be considered as first-tier diagnostics in NDDE. This high yield and the numerous indications for additional screening or treatment modifications arising from genetic diagnoses indicate a current medical undersupply of genetically undiagnosed adult/elderly individuals with NDDE. Moreover, knowledge of the course of elderly individuals will ultimately help in counseling newly diagnosed individuals with NDDE.

Here we report a heterozygous tandem duplication at the ASIP (agouti signaling protein) gene locus causing ubiquitous, ectopic ASIP expression in a female patient with extreme childhood obesity. The mutation places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase promoter, driving the generation of ASIP in patient-derived native and induced pluripotent stem cells for all germ layers and hypothalamic-like neurons. The patient’s phenotype of early-onset obesity, overgrowth, red hair and hyperinsulinemia is concordant with that of mutant mice ubiquitously expressing the homolog nonagouti. ASIP represses melanocyte-stimulating hormone-mediated activation as a melanocortin receptor antagonist, which might affect eating behavior, energy expenditure, adipocyte differentiation and pigmentation, as observed in the index patient. As the type of mutation escapes standard genetic screening algorithms, we rescreened the Leipzig Childhood Obesity cohort of 1,745 patients and identified four additional patients with the identical mutation, ectopic ASIP expression and a similar phenotype. Taken together, our data indicate that ubiquitous ectopic ASIP expression is likely a monogenic cause of human obesity.

Background: Little is known so far about the brain phenotype and the spatial interplay of different Alzheimer’s disease (AD) biomarkers with structural and functional brain connectivity in the early phase of autosomal-dominant AD (ADAD). Multimodal PET/MRI might be suitable to fill this gap. Material and Methods: We presented a 31-year-old male patient without a family history of dementia with progressive worsening of memory and motor function. Two separate sessions of 3T PET/MRI acquisitions were arranged with the ß-amyloid tracer [18F]Florbetaben and the secondgeneration tau tracer [18F]PI-2620. Simultaneously acquired MRI consisted of high-resolution 3D T1, diffusion-tensor imaging (DTI), and resting-state fMRI. PET/MRI data were compared with ten age-matched healthy controls. Results: Widespread β-amyloid depositions were found in cortical regions, and striatum (Thal stage III) along with tau pathology restricted to the mesial-temporal structures (Braak stage III/IV). Volumetric/shape analysis of subcortical structures revealed atrophy of the hippocampal-amygdala complex. In addition, cortical thinning was detected in the right middle temporal pole. Alterations of multiple DTI indices were noted in the major white matter fiber bundles, together with disruption of default mode and sensory-motor network functional connectivity. Molecular genetic analysis by next-generation sequencing revealed a heterozygote missense pathogenic variant of the PSEN1 (Met233Val). Conclusion: Multimodal PET/MR imaging is able to deliver, in a one-stop-shop approach, an array of molecular, structural and functional brain information in AD due to de novo pathogenic variant, which can be studied for spatial interplay and might provide a rationale for initiating anti- amyloid/tau therapeutic approaches.

PURPOSE Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy (DEE) with early-onset seizures and severe intellectual disability. METHODS By international collaboration we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders (NDDs). By western blotting we investigated the consequences of missense variants in vitro. RESULTS In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe DEE in 16 individuals. We now identified also de novo missense variants in the GTPase domain in six individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences.Furthermore, we observed bi-allelic splice-site and truncating variants in nine families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well. CONCLUSION By identifying phenotype-genotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying bi-allelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2 related disorders including both autosomal dominant and recessive NDDs.

RIPPLY2 is an essential part of the formation of somite patterning during embryogenesis and in establishment of rostro-caudal polarity. Here, we describe three individuals from two families with compound-heterozygous variants in RIPPLY2 (NM_001009994.2): c.238A > T, p.(Arg80*) and c.240-4 T > G, p.(?), in two 15 and 20 year old sisters, and a homozygous nonsense variant, c.238A > T, p.(Arg80*), in an 8 year old boy. All patients had multiple vertebral body malformations in the cervical and thoracic region, small or absent rib involvement, myelopathies, and common clinical features of SCDO6 including scoliosis, mild facial asymmetry, spinal spasticity and hemivertebrae. The nonsense variant can be classified as likely pathogenic based on the ACMG criteria while the splice variants must be classified as a variant of unknown significance. With this report on two further families, we confirm RIPPLY2 as the gene for SCDO6 and broaden the phenotype by adding myelopathy with or without spinal canal stenosis and spinal spasticity to the symptom spectrum. This article is protected by copyright. All rights reserved.

Routine exome sequencing (ES) in individuals with neurodevelopmental disorders (NDD) remains inconclusive in >50%. Research analysis of unsolved cases can identify novel candidate genes but is time consuming, subjective, and hard to compare between labs. The field therefore needs automated and standardized assessment methods to prioritize candidates for matchmaking. We developed AutoCaSc (https://autocasc.uni-leipzig.de) based on our candidate scoring scheme (CaSc). We validated our approach using synthetic trios and real in-house trio ES data. AutoCaSc consistently (94.5%) scored variants in valid novel NDD genes in the top three ranks. In 93 real trio exomes, AutoCaSc identified most (97.5%) previously manually scored variants while evaluating additional highly scoring variants missed in manual evaluation. It identified candidate variants in previously undescribed NDD candidate genes ( CNTN2, DLGAP1, SMURF1, NRXN3, PRICKLE1). AutoCaSc enables anybody to quickly screen a variant for its plausibility in NDD. After contributing >40 descriptions of NDD associated genes, we provide usage recommendations based on our extensive experience. Our implementation is capable of pipeline integration and therefore allows screening of large cohorts for candidate genes. AutoCaSc empowers even small labs to a standardized matchmaking collaboration and to contribute to the ongoing identification of novel NDD entities.

The endocannabinoid system is a highly conserved and ubiquitous signalling pathway with broad-ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNA sequencing showed clear expression of the truncated transcript and no differences were found between mutant and wild-type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique paediatric syndrome. Because enzymatic activity was preserved, the observed mislocalization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues.

PPFIBP1 encodes for the liprin-β1 protein which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 14 individuals from 10 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy and progressive microcephaly. Further common clinical findings included muscular hypertonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired hearing and vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM-domain region that is essential for protein-protein interaction. For further insight in the effects of PPFIBP1 loss-of-function, we performed automated behavioural phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model which revealed defects in spontaneous and light-induced behaviour and confirmed resistance to the acetylcholinesterase inhibitor aldicarb suggesting a defect in the neuronal presynaptic zone. In conclusion, we present bi-allelic loss-of-function variants in PPFIBP1 as a novel cause of an autosomal recessive neurodevelopmental disorder.

Pathogenic variants inTP53have been classically thought to cause Li-Fraumeni syndrome (LFS), a cancer predisposition with high risks for various childhood- and adult-onset malignancies. However, increased genetic testing has lately revealed, that pathogenic variant carriers exhibit a broader range of phenotypes and that penetrance may be dependent both on variant type and modifiers. Using next generation sequencing and short tandem repeat analysis, we identified germline pathogenic variants inTP53andRAD51Clocated incison chromosome 17 in a 43-year-old male, who has developed a rare sebaceous gland carcinoma (SGC) but so far no tumors of the LFS spectrum. This course mirrors aTrp53-Rad51c-double-mutantcismouse-model, which similarly develops SGC, while the characteristicTrp53-associated tumor spectrum occurs with significantly lower frequency. Therefore, we propose that co-occurent pathogenic variants inRAD51CandTP53may predispose to SGC, reminiscent of Muir-Torre syndrome. Further, this report supports the diversity of clinical presentations associated with germlineTP53alterations, and thus, the proposed expansion of LFS to heritableTP53-related cancer syndrome.

KCNN2 encodes the small conductance calcium-activated potassium channel 2 (SK2). Rodent models with spontaneous Kcnn2 mutations show abnormal gait and locomotor activity, tremor and memory deficits, but human disorders related to KCNN2 variants are largely unknown. Using exome sequencing, we identified a de novo KCNN2 frameshift deletion in a patient with learning disabilities, cerebellar ataxia and white matter abnormalities on brain MRI. This discovery prompted us to collect data from nine additional patients with de novo KCNN2 variants (one nonsense, one splice site, six missense variants and one in-frame deletion) and one family with a missense variant inherited from the affected mother. We investigated the functional impact of six selected variants on SK2 channel function using the patch-clamp technique. All variants tested but one, which was reclassified to uncertain significance, led to a loss-of-function of SK2 channels. Patients with KCNN2 variants had motor and language developmental delay, intellectual disability often associated with early-onset movement disorders comprising cerebellar ataxia and/or extrapyramidal symptoms. Altogether, our findings provide evidence that heterozygous variants, likely causing a haploinsufficiency of the KCNN2 gene, lead to novel autosomal dominant neurodevelopmental movement disorders mirroring phenotypes previously described in rodents.

Objective: Autosomal recessive pathogenic variants of the SLC13A5 gene are associated with severe neonatal epilepsy, developmental delay, and tooth hypoplasia/hypodontia. We report on 14 additional patients and compare their phenotypic features to previously published patients to identify the clinical hallmarks of this disorder. Methods: We collected clinical features of 14 patients carrying biallelic variants in SLC13A5 and performed a PubMed search to identify previously published patients. Results: All patients presented clonic or tonic seizures in the first days of life, evolving into status epilepticus in 57%. Analysis of seizure frequency and developmental milestones divided into five epochs showed an evolutionary trajectory of both items. In the first 3 years of life, 72% of patients had weekly/monthly seizures, often triggered by fever; 14% were seizure-free. Between the ages of 3 and 12 years, 60% become seizure-free; in the following years, up to age 18 years, 57% were seizure-free. After the age of 18 years, all three patients reaching this age were seizure-free. Similarly, 86% of patients at onset presented mild to moderate developmental impairment and diffuse hypotonia. In late childhood, all had developmental delay that was severe in most. Benzodiazepines, phenobarbital, phenytoin, and carbamazepine were the most effective drugs. Eight probands carried heterozygous compound variants, and homozygous pathogenic variants occurred in six. Literature review identified 45 patients carrying SLC13A5 gene pathogenic variants whose clinical features overlapped with our cohort. A peculiar and distinguishing sign is the presence of tooth hypoplasia and/or hypodontia in most patients. Significance: Autosomal recessive pathogenic variants in SLC13A5 are associated with a distinct neonatal epileptic encephalopathy evolving into severe cognitive and motor impairment, yet with seizures that settle down in late childhood. Tooth hypoplasia or hypodontia remains the peculiar feature. The SLC13A5 gene should be screened in neonatal epileptic encephalopathies; its recessive inheritance has relevance for genetic counseling.

In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.

In a patient with extreme childhood obesity, we identified a heterozygous tandem duplication at the ASIP (agouti-signaling protein) gene locus causing ubiquitous, ectopic ASIP expression. The mutation places ASIP under control of the ubiquitously active itchy E3 ubiquitin protein ligase (ITCH) promoter, driving the ubiquitous generation of ASIP as evidenced in patient-derived induced pluripotent stemm cells for all germ layers. The patient´s phenotype of early-onset obesity, overgrowth, red hair, and hyperinsulinemia is concordant with that of mutant mice ubiquitously expressing the homolog agouti. ASIP represses melanocyte-stimulating hormone-mediated activation of as an inverse agonist of the melanocortin receptors, thereby affecting eating behavior, energy expenditure, adipocyte differentiation, and pigmentation, as observed in the index patient. Furthermore, we identified another patient with the identical mutation, ectopic ASIP expression and a similar phenotype. Thus, human obesity caused by ubiquitous ASIP expression is a novel monogenic trait, potentially treatable by melanocortin receptor agonists.

PurposeNSD2 dimethylates histone H3 at lysine 36 (H3K36me2) and is located in the Wolf-Hirschhorn syndrome (WHS) region. Recent descriptions delineated loss-of-function (LoF) variants in NSD2 with a distinct disorder. The oncogenic missense variant p.Glu1099Lys occures somatically in leukemia and has a gain-of-function (GoF) effect.MethodsWe describe two unrelated individuals carrying c.3295G>A, p.Glu1099Lys as heterozygous de novo germline variants identified by exome sequencing of blood DNA and subsequently confirmed in two ectodermal tissues. We use omics data from the Cancer Cell Line Encyclopedia to analyze the GoF effect.ResultsClinically these individuals are characterized by intellectual disability, coarse/ square facial gestalt, abnormalities of the hands and organomegaly. We confirmed increased K36me2 methylation in lines with either NSD2 GoF variants or duplications. Cells with GoF variants showed increased DNA promoter methylation and dysregulated RNA expression, influencing cellular modules involved in white blood cell activation, cell growth and organ development.ConclusionNSD2 GoF caused by the missense variant p.Glu1099Lys leads to a novel syndromic phenotype distinct from both the previously described LoF phenotypes. Other variants causing NSD2 hyperactivation or overexpression may cause a similar phenotype. This syndrome should be distinguished from the recently named Rauch-Steindl syndrome caused by NSD2 haploinsufficiency.

Hypochondroplasia is a skeletal dysplasia syndrome with an autosomal dominant inheritance. It may be associated with temporal lobe epilepsy. We present a series of four patients (two female, two male) with hypochondroplasia who presented at our center with drug refractory epilepsy. Clinical details and EEG and MRI findings led to a diagnosis of temporal lobe epilepsy in all four cases. The MRI findings indicate the epilepsy in hypochondroplasia may be associated with bilateral temporal lobe dysgenesis. Copyright © 2021 Elsevier Inc. All rights reserved.

Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.