Genome sequencing and comprehensive rare-variant analysis of 465 families with neurodevelopmental disorders.

Despite significant progress in unraveling the genetic causes of neurodevelopmental disorders (NDDs), a substantial proportion of individuals with NDDs remain without a genetic diagnosis after microarray and/or exome sequencing. Here, we aimed to assess the power of short-read genome sequencing (GS), complemented with long-read GS, to identify causal variants in participants with NDD from the National Institute for Health and Care Research (NIHR) BioResource project. Short-read GS was conducted on 692 individuals (489 affected and 203 unaffected relatives) from 465 families. Additionally, long-read GS was performed on five affected individuals who had structural variants (SVs) in technically challenging regions, had complex SVs, or required distal variant phasing. Causal variants were identified in 36% of affected individuals (177/489), and a further 23% (112/489) had a variant of uncertain significance after multiple rounds of re-analysis. Among all reported variants, 88% (333/380) were coding nuclear SNVs or insertions and deletions (indels), and the remainder were SVs, non-coding variants, and mitochondrial variants. Furthermore, long-read GS facilitated the resolution of challenging SVs and invalidated variants of difficult interpretation from short-read GS. This study demonstrates the value of short-read GS, complemented with long-read GS, in investigating the genetic causes of NDDs. GS provides a comprehensive and unbiased method of identifying all types of variants throughout the nuclear and mitochondrial genomes in individuals with NDD.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

De Novo Truncating Mutations in WASF1 Cause Intellectual Disability with Seizures.

Next-generation sequencing has been invaluable in the elucidation of the genetic etiology of many subtypes of intellectual disability in recent years. Here, using exome sequencing and whole-genome sequencing, we identified three de novo truncating mutations in WAS protein family member 1 (WASF1) in five unrelated individuals with moderate to profound intellectual disability with autistic features and seizures. WASF1, also known as WAVE1, is part of the WAVE complex and acts as a mediator between Rac-GTPase and actin to induce actin polymerization. The three mutations connected by Matchmaker Exchange were c.1516C>T (p.Arg506Ter), which occurs in three unrelated individuals, c.1558C>T (p.Gln520Ter), and c.1482delinsGCCAGG (p.Ile494MetfsTer23). All three variants are predicted to partially or fully disrupt the C-terminal actin-binding WCA domain. Functional studies using fibroblast cells from two affected individuals with the c.1516C>T mutation showed a truncated WASF1 and a defect in actin remodeling. This study provides evidence that de novo heterozygous mutations in WASF1 cause a rare form of intellectual disability.

Impairment of adenosinergic system in Rett syndrome: Novel therapeutic target to boost BDNF signalling.

Rett syndrome (RTT; OMIM#312750) is mainly caused by mutations in the X-linked MECP2 gene (methyl-CpG-binding protein 2 gene; OMIM*300005), which leads to impairments in the brain-derived neurotrophic factor (BDNF) signalling. The boost of BDNF mediated effects would be a significant breakthrough but it has been hampered by the difficulty to administer BDNF to the central nervous system. Adenosine, an endogenous neuromodulator, may accomplish that role since through A2AR it potentiates BDNF synaptic actions in healthy animals. We thus characterized several hallmarks of the adenosinergic and BDNF signalling in RTT and explored whether A2AR activation could boost BDNF actions. For this study, the RTT animal model, the Mecp2 knockout (Mecp2-/y) (B6.129P2 (C)-Mecp2tm1.1Bird/J) mouse was used. Whenever possible, parallel data was also obtained from post-mortem brain samples from one RTT patient. Ex vivo extracellular recordings of field excitatory post-synaptic potentials in CA1 hippocampal area were performed to evaluate synaptic transmission and long-term potentiation (LTP). RT-PCR was used to assess mRNA levels and Western Blot or radioligand binding assays were performed to evaluate protein levels. Changes in cortical and hippocampal adenosine content were assessed by liquid chromatography with diode array detection (LC/DAD). Hippocampal ex vivo experiments revealed that the facilitatory actions of BDNF upon LTP is absent in Mecp2-/y mice and that TrkB full-length (TrkB-FL) receptor levels are significantly decreased. Extracts of the hippocampus and cortex of Mecp2-/y mice revealed less adenosine amount as well as less A2AR protein levels when compared to WT littermates, which may partially explain the deficits in adenosinergic tonus in these animals. Remarkably, the lack of BDNF effect on hippocampal LTP in Mecp2-/y mice was overcome by selective activation of A2AR with CGS21680. Overall, in Mecp2-/y mice there is an impairment on adenosinergic system and BDNF signalling. These findings set the stage for adenosine-based pharmacological therapeutic strategies for RTT, highlighting A2AR as a therapeutic target in this devastating pathology.

Loss of hierarchical imprinting regulation at the Prader-Willi/Angelman syndrome locus in human iPSCs.

The human chr15q11-q13 imprinted cluster is linked to several disorders, including Prader-Willi (PWS) and Angelman (AS) syndromes. Recently, disease modeling approaches based on induced pluripotent stem cells (iPSCs) have been used to study these syndromes. A concern regarding the use of these cells for imprinted disease modeling is the numerous imprinting defects found in many iPSCs. Here, by reprogramming skin fibroblasts from a control and AS individuals, we generated several iPSC lines and addressed the stability of imprinting status across the PWS/AS domain. We focused on three important regulatory DNA elements which are all differentially methylated regions (DMRs), methylated on the maternal allele: the PWS imprinting center (PWS-IC), which is a germline DMR and the somatic NDN and MKRN3 DMRs, hierarchically controlled by PWS-IC. Normal PWS-IC methylation pattern was maintained in most iPSC lines; however, loss of maternal methylation in one out of five control iPSC lines resulted in a monoallelic to biallelic switch for many imprinted genes in this domain. Surprisingly, MKRN3 DMR was found aberrantly hypermethylated in all control and AS iPSCs, regardless of the methylation status of the PWS-IC master regulator. This suggests a loss of hierarchical control of imprinting at PWS/AS region. We confirmed these results in established iPSC lines derived using different reprogramming procedures. Overall, we show that hierarchy of imprinting control in donor cells might not apply to iPSCs, accounting for their spectrum of imprinting alterations. Such differences in imprinting regulation should be taken into consideration for the use of iPSCs in disease modeling.

Adenosine A1 Receptor Suppresses Tonic GABAA Receptor Currents in Hippocampal Pyramidal Cells and in a Defined Subpopulation of Interneurons.

Adenosine is an endogenous neuromodulator that decreases excitability of hippocampal circuits activating membrane-bound metabotropic A1 receptor (A1R). The presynaptic inhibitory action of adenosine A1R in glutamatergic synapses is well documented, but its influence on inhibitory GABAergic transmission is poorly known. We report that GABAA receptor (GABAAR)-mediated tonic, but not phasic, transmission is suppressed by A1R in hippocampal neurons. Adenosine A1R activation strongly inhibits GABAAR agonist (muscimol)-evoked currents in Cornu Ammonis 1 (CA1) pyramidal neurons and in a specific subpopulation of interneurons expressing axonal cannabinoid receptor type 1. In addition, A1R suppresses tonic GABAAR currents measured in the presence of elevated ambient GABA as well as in naïve slices. The inhibition of GABAergic currents involves both protein kinase A (PKA) and protein kinase C (PKC) signaling pathways and decreases GABAAR δ-subunit expression. On the contrary, no A1R-mediated modulation was detected in phasic inhibitory postsynaptic currents evoked either by afferent electrical stimulation or by spontaneous quantal release. The results show that A1R modulates extrasynaptic rather than synaptic GABAAR-mediated signaling, and that this modulation selectively occurs in hippocampal pyramidal neurons and in a specific subpopulation of inhibitory interneurons. We conclude that modulation of tonic GABAAR signaling by adenosine A1R in specific neuron types may regulate neuronal gain and excitability in the hippocampus.

Dominant and recessive RYR1 mutations in adults with core lesions and mild muscle symptoms.

INTRODUCTION: Ryanodine receptor gene (RYR1) mutations have been associated with central core disease (CCD), multiminicore/minicore/multicore disease (MmD), and susceptibility to malignant hyperthermia (MH). METHODS: Patients with muscle symptoms in adulthood, who had features compatible with CCD/MmD, underwent clinical, histological, and genetic (RYR1 and SEPN1 genes) evaluations. Published cases of CCD and MmD with adult onset were also reviewed. RESULTS: Eight patients fulfilled the criteria for further analysis. Five RYR1 mutations, 4 of them unreported, were detected in 3 patients. Compound heterozygosity was proven in 1 case. CONCLUSIONS: To our knowledge, this is the only report of adult onset associated with recessive RYR1 mutations and central core/multiminicores on muscle biopsy. Although adult patients with CCD, MmD, and minimally symptomatic MH with abnormal muscle biopsy findings usually have a mild clinical course, differential diagnosis and carrier screening is crucial for prevention of potentially life-threatening reactions to general anesthesia.

Analysis of synaptic proteins in the cerebrospinal fluid as a new tool in the study of inborn errors of neurotransmission.

In a few rare diseases, specialised studies in cerebrospinal fluid (CSF) are required to identify the underlying metabolic disorder. We aimed to explore the possibility of detecting key synaptic proteins in the CSF, in particular dopaminergic and gabaergic, as new procedures that could be useful for both pathophysiological and diagnostic purposes in investigation of inherited disorders of neurotransmission. Dopamine receptor type 2 (D2R), dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2) were analysed in CSF samples from 30 healthy controls (11 days to 17 years) by western blot analysis. Because VMAT2 was the only protein with intracellular localisation, and in order to compare results, GABA vesicular transporter, which is another intracellular protein, was also studied. Spearman's correlation and Student's t tests were applied to compare optical density signals between different proteins. All these synaptic proteins could be easily detected and quantified in the CSF. DAT, D2R and GABA VT expression decrease with age, particularly in the first months of life, reflecting the expected intense synaptic activity and neuronal circuitry formation. A statistically significant relationship was found between D2R and DAT expression, reinforcing the previous evidence of DAT regulation by D2R. To our knowledge, there are no previous studies on human CSF reporting a reliable analysis of these proteins. These kinds of studies could help elucidate new causes of disturbed dopaminergic and gabaergic transmission as well as understanding different responses to L-dopa in inherited disorders affecting dopamine metabolism. Moreover, this approach to synaptic activity in vivo can be extended to different groups of proteins and diseases.

Structural analysis of pathogenic missense mutations in GABRA2 and identification of a novel de novo variant in the desensitization gate.

BACKGROUND: Cys-loop receptors control neuronal excitability in the brain and their dysfunction results in numerous neurological disorders. Recently, six missense variants in GABRA2, a member of this family, have been associated with early infantile epileptic encephalopathy (EIEE). We identified a novel de novo missense variant in GABRA2 in a patient with EIEE and performed protein structural analysis of the seven variants. METHODS: The novel variant was identified by trio whole-genome sequencing. We performed protein structural analysis of the seven variants, and compared them to previously reported pathogenic mutations at equivalent positions in other Cys-loop receptors. Additionally, we studied the distribution of disease-associated variants in the transmembrane helices of these proteins. RESULTS: The seven variants are in the transmembrane domain, either close to the desensitization gate, the activation gate, or in inter-subunit interfaces. Six of them have pathogenic mutations at equivalent positions in other Cys-loop receptors, emphasizing the importance of these residues. Also, pathogenic mutations are more common in the pore-lining helix, consistent with this region being highly constrained for variation in control populations. CONCLUSION: Our study reports a novel pathogenic variant in GABRA2, characterizes the regions where pathogenic mutations are in the transmembrane helices, and underscores the value of considering sequence, evolutionary, and structural information as a strategy for variant interpretation of novel missense mutations.

Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells.

Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms.

Atypical phenotype in two patients with LAMA2 mutations.

Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene, which encodes the α2-chain of laminin. We report two patients with partial laminin-α2 deficiency and atypical phenotypes, one with almost exclusive central nervous system involvement (cognitive impairment and refractory epilepsy) and the second with marked cardiac dysfunction, rigid spine syndrome and limb-girdle weakness. Patients underwent clinical, histopathological, imaging and genetic studies. Both cases have two heterozygous LAMA2 variants sharing a potentially pathogenic missense mutation c.2461A>C (p.Thr821Pro) located in exon 18. Brain MRI was instrumental for the diagnosis, since muscular examination and motor achievements were normal in the first patient and there was a severe cardiac involvement in the second. The clinical phenotype of the patients is markedly different which could in part be explained by the different combination of mutations types (two missense versus a missense and a truncating mutation).

Undetectable levels of CSF amyloid-ß peptide in a patient with 17ß-hydroxysteroid dehydrogenase deficiency.

17ß-hydroxysteroid dehydrogenase 10 (HSD10) deficiency is a rare X-linked inborn error of isoleucine catabolism. Although this protein has been genetically implicated in Alzheimer's disease pathogenesis, studies of amyloid-ß peptide (Aß) in patients with HSD10 deficiency have not been previously reported. We found, in a severely affected child with HSD10 deficiency, undetectable levels of Aß in the cerebrospinal fluid, together with low expression of brain-derived neurotrophic factor, α-synuclein, and serotonin metabolites. Confirmation of these findings in other patients would help elucidating mechanisms of synaptic dysfunction in this disease, and highlight the role of Aß in both early and late periods of life.