A cross-species proteomic map reveals neoteny of human synapse development.

The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

Limb girdle muscular disease caused by HMGCR mutation and statin myopathy treatable with mevalonolactone.

Myopathy is the main adverse effect of the widely prescribed statin drug class. Statins exert their beneficial effect by inhibiting HMG CoA-reductase, the rate-controlling enzyme of the mevalonate pathway. The mechanism of statin myopathy is yet to be resolved, and its treatment is insufficient. Through homozygosity mapping and whole exome sequencing, followed by functional analysis using confocal microscopy and biochemical and biophysical methods, we demonstrate that a distinct form of human limb girdle muscular disease is caused by a pathogenic homozygous loss-of-function missense mutation in HMG CoA reductase (HMGCR), encoding HMG CoA-reductase. We biochemically synthesized and purified mevalonolactone, never administered to human patients before, and establish the safety of its oral administration in mice. We then show that its oral administration is effective in treating a human patient with no significant adverse effects. Furthermore, we demonstrate that oral mevalonolactone resolved statin-induced myopathy in mice. We conclude that HMGCR mutation causes a late-onset severe progressive muscular disease, which shows similar features to statin-induced myopathy. Our findings indicate that mevalonolactone is effective both in the treatment of hereditary HMGCR myopathy and in a murine model of statin myopathy. Further large clinical trials are in place to enable the clinical use of mevalonolactone both in the rare orphan disease and in the more common statin myopathy.

ZNF142 mutation causes sex-dependent neurologic disorder.

BACKGROUND: Sex-specific predilection in neurological diseases caused by mutations in autosomal genes is a phenomenon whose molecular basis is poorly understood. We studied females of consanguineous Bedouin kindred presenting with severe global developmental delay and epilepsy. METHODS: Linkage analysis, whole exome sequencing, generation of CRISPR/cas9 knock-in mice, mouse behaviour and molecular studies RESULTS: Linkage analysis and whole exome sequencing studies of the affected kindred delineated a ~5 Mbp disease-associated chromosome 2q35 locus, containing a novel homozygous frameshift truncating mutation in ZNF142, in line with recent studies depicting similar ZNF142 putative loss-of-function human phenotypes with female preponderance. We generated knock-in mice with a truncating mutation adjacent to the human mutation in the mouse ortholog. Behaviour studies of homozygous Zfp142R1508* mice showed significant phenotype only in mutant females, with learning and memory deficits, hyperactivity and aberrant loss of fear of open spaces. Bone marrow and spleen of homozygous Zfp142R1508* mice showed depletion of lymphoid and haematopoietic cells, mostly in females. RT-PCR showed lower expression of Zpf142 in brain compartments of female versus male wild-type mice. RNA-seq studies of hippocampus, hypothalamus, cortex and cerebellum of female wild-type versus homozygous Zfp142R1508* mice demonstrated differentially expressed genes. Notably, expression of Taok1 in the cortex and of Mllt6 in the hippocampus was downregulated in homozygous Zfp142R1508* mice. Taok1 mutations have been associated with aberrant neurodevelopment and behaviour. Mllt6 expression is regulated by sex hormones and Mllt6 null-mutant mice present with haematopoietic, immune system and female-specific behaviour phenotypes. CONCLUSION: ZNF142 mutation downregulates Mllt6 and Taok1, causing a neurodevelopmental phenotype in humans and mice with female preponderance.

Tropism of SARS-CoV-2 for human cortical astrocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Factors for success of awake prone positioning in patients with COVID-19-induced acute hypoxemic respiratory failure: analysis of a randomized controlled trial.

BACKGROUND: Awake prone positioning (APP) improves oxygenation in coronavirus disease (COVID-19) patients and, when successful, may decrease the risk of intubation. However, factors associated with APP success remain unknown. In this secondary analysis, we aimed to assess whether APP can reduce intubation rate in patients with COVID-19 and to focus on the factors associated with success. METHODS: In this multicenter randomized controlled trial, conducted in three high-acuity units, we randomly assigned patients with COVID-19-induced acute hypoxemic respiratory failure (AHRF) requiring high-flow nasal cannula (HFNC) oxygen to APP or standard care. Primary outcome was intubation rate at 28 days. Multivariate analyses were performed to identify the predictors associated to treatment success (survival without intubation). RESULTS: Among 430 patients randomized, 216 were assigned to APP and 214 to standard care. The APP group had a lower intubation rate (30% vs 43%, relative risk [RR] 0.70; CI95 0.54-0.90, P = 0.006) and shorter hospital length of stay (11 interquartile range [IQR, 9-14] vs 13 [IQR, 10-17] days, P = 0.001). A respiratory rate ≤ 25 bpm at enrollment, an increase in ROX index > 1.25 after first APP session, APP duration > 8 h/day, and a decrease in lung ultrasound score ≥ 2 within the first 3 days were significantly associated with treatment success for APP. CONCLUSION: In patients with COVID-19-induced AHRF treated by HFNC, APP reduced intubation rate and improved treatment success. A longer APP duration is associated with APP success, while the increase in ROX index and decrease in lung ultrasound score after APP can also help identify patients most likely to benefit. TRIAL REGISTRATION: This study was retrospectively registered in ClinicalTrials.gov at July 20, 2021. Identification number NCT04477655. https://clinicaltrials.gov/ct2/show/NCT04477655?term=PRO-CARF&draw=2&rank=1.

Mutations in the microtubule-associated protein MAP11 (C7orf43) cause microcephaly in humans and zebrafish.

Microtubule associated protein 11 (MAP11, previously termed C7orf43) encodes a highly conserved protein whose function is unknown. Through genome-wide linkage analysis combined with whole exome sequencing, we demonstrate that human autosomal recessive primary microcephaly is caused by a truncating mutation in MAP11. Moreover, homozygous MAP11-orthologue CRISPR/Cas9 knock-out zebrafish presented with microcephaly and decreased neuronal proliferation, recapitulating the human phenotype. We demonstrate that MAP11 is ubiquitously transcribed with high levels in brain and cerebellum. Immunofluorescence and co-immunoprecipitation studies in SH-SY5Y cells showed that MAP11 associates with mitotic spindles, co-localizing and physically associating with α-tubulin during mitosis. MAP11 expression precedes α-tubulin in gap formation of cell abscission at the midbody and is co-localized with PLK1, a key regulator of cytokinesis, at the edges of microtubule extensions of daughter cells post cytokinesis abscission, implicating a role in mitotic spindle dynamics and in regulation of cell abscission during cytokinesis. Finally, lentiviral-mediated silencing of MAP11 diminished SH-SY5Y cell viability, reducing proliferation rather than affecting apoptosis. Thus, MAP11 encodes a microtubule-associated protein that plays a role in spindle dynamics and cell division, in which mutations cause microcephaly in humans and zebrafish.

SEC31A mutation affects ER homeostasis, causing a neurological syndrome.

BACKGROUND: Consanguineous kindred presented with an autosomal recessive syndrome of intrauterine growth retardation, marked developmental delay, spastic quadriplegia with profound contractures, pseudobulbar palsy with recurrent aspirations, epilepsy, dysmorphism, neurosensory deafness and optic nerve atrophy with no eye fixation. Affected individuals died by the age of 4. Brain MRI demonstrated microcephaly, semilobar holoprosencephaly and agenesis of corpus callosum. We aimed at elucidating the molecular basis of this disease. METHODS: Genome-wide linkage analysis combined with whole exome sequencing were performed to identify disease-causing variants. Functional consequences were investigated in fruit flies null mutant for the Drosophila SEC31A orthologue. SEC31A knockout SH-SY5Y and HEK293T cell-lines were generated using CRISPR/Cas9 and studied through qRT-PCR, immunoblotting and viability assays. RESULTS: Through genetic studies, we identified a disease-associated homozygous nonsense mutation in SEC31A. We demonstrate that SEC31A is ubiquitously expressed, and that the mutation triggers nonsense-mediated decay of its transcript, comprising a practical null mutation. Similar to the human disease phenotype, knockdown SEC31A flies had defective brains and early lethality. Moreover, in line with SEC31A encoding one of the two coating layers comprising the Coat protein complex II (COP-II) complex, trafficking newly synthesised proteins from the endoplasmic reticulum (ER) to the Golgi, CRISPR/Cas9-mediated SEC31A null mutant cells demonstrated reduced viability through upregulation of ER-stress pathways. CONCLUSION: We demonstrate through human and Drosophila genetic and in vitro molecular studies, that a severe neurological syndrome is caused by a null mutation in SEC31A, reducing cell viability through enhanced ER-stress response, in line with SEC31A's role in the COP-II complex.

RSRC1 mutation affects intellect and behaviour through aberrant splicing and transcription, downregulating IGFBP3.

RSRC1, whose polymorphism is associated with altered brain function in schizophrenia, is a member of the serine and arginine rich-related protein family. Through homozygosity mapping and whole exome sequencing we show that RSRC1 mutation causes an autosomal recessive syndrome of intellectual disability, aberrant behaviour, hypotonia and mild facial dysmorphism with normal brain MRI. Further, we show that RSRC1 is ubiquitously expressed, and that the RSRC1 mutation triggers nonsense-mediated mRNA decay of the RSRC1 transcript in patients' fibroblasts. Short hairpin RNA (shRNA)-mediated lentiviral silencing and overexpression of RSRC1 in SH-SY5Y cells demonstrated that RSRC1 has a role in alternative splicing and transcription regulation. Transcriptome profiling of RSRC1-silenced cells unravelled specific differentially expressed genes previously associated with intellectual disability, hypotonia and schizophrenia, relevant to the disease phenotype. Protein-protein interaction network modelling suggested possible intermediate interactions by which RSRC1 affects gene-specific differential expression. Patient-derived induced pluripotent stem cells, differentiated into neural progenitor cells, showed expression dynamics similar to the RSRC1-silenced SH-SY5Y model. Notably, patient neural progenitor cells had 9.6-fold downregulated expression of IGFBP3, whose brain expression is affected by MECP2, aberrant in Rett syndrome. Interestingly, Igfbp3-null mice have behavioural impairment, abnormal synaptic function and monoaminergic neurotransmission, likely correlating with the disease phenotype.

ALFY-Controlled DVL3 Autophagy Regulates Wnt Signaling, Determining Human Brain Size.

Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size.

A novel homozygous SLC25A1 mutation with impaired mitochondrial complex V: Possible phenotypic expansion.

SLC25A1 mutations are associated with combined D,L-2-hydroxyglutaric aciduria (DL- 2HGA; OMIM #615182), characterized by muscular hypotonia, severe neurodevelopmental dysfunction and intractable seizures. SLC25A1 encodes the mitochondrial citrate carrier (CIC), which mediates efflux of the mitochondrial tricarboxylic acid (TCA) cycle intermediates citrate and isocitrate in exchange for cytosolic malate. Only a single family with an SLC25A1 mutation has been described in which mitochondrial respiratory chain dysfunction was documented, specifically in complex IV. Five infants of two consanguineous Bedouin families of the same tribe presented with small head circumference and neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development culminating in early death. Ventricular septal defects (VSD) were demonstrated in three patients. Blood and CSF lactate were elevated with normal levels of plasma amino acids and free carnitine and increased 2-OH-glutaric acid urinary exertion. EEG was compatible with white matter disorder. Brain MRI revealed ventriculomegaly, thin corpus callosum with increased lactate peak on spectroscopy. Mitochondrial complex V deficiency was demonstrated in skeletal muscle biopsy of one infant. Homozygosity mapping and sequencing ruled out homozygosity of affected individuals in all known complex V-associated genes. Whole exome sequencing identified a novel homozygous SLC25A1 c.713A>G (p.Asn238Ser) mutation, segregating as expected in the affected kindred and not found in 220 control alleles. Thus, SLC25A1 mutations might be associated with mitochondrial complex V deficiency and should be considered in the differential diagnosis of mitochondrial respiratory chain defects.

Combined CNV, haplotyping and whole exome sequencing enable identification of two distinct novel EYS mutations causing RP in a single inbred tribe.

Whole exome sequencing (WES) has become routine in clinical practice, especially in studies of recessive hereditary diseases in inbred consanguineous families, where homozygosity of a founder mutation is assumed. Multiple members of two consanguineous families of a single Bedouin tribe were diagnosed with apparently autosomal recessive/pseudo-dominant retinitis pigmentosa (RP). Affected individuals exhibited severe visual impairment with nyctalopia, marked constriction of visual fields, markedly reduced and delayed responses on electro-retinography (ERG) and eventual loss of central vision. Combined copy-number variant (CNV) analysis, haplotype reconstruction and WES of the kindred identified two distinct novel mutations in EYS (RP25): a p.(W1817*) nonsense mutation (identified through WES) and a large deletion encompassing 9 of the 43 exons, that was missed by WES and was identified through microarray CNV analysis. Segregation analysis of both mutations demonstrated that all affected individuals were either homozygous for one of the mutations, or compound heterozygous for both. The two mutations are predicted to cause loss of function of the encoded protein and were not present in screening of 200 ethnically-matched controls. Our findings of two distinct mutations in the same gene in a single inbred kindred, identified only through combined WES and microarray CNV analysis, highlight the limitations of either CNV or WES alone, as the heterozygous deletion had normal WES read-depth values. Moreover, they demonstrate pitfalls in homozygosity mapping for disease-causing variant identification in inbred communities.

SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome.

A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through defective function of this novel activity of SLC30A9 rather than by a defect in its previously described role in transcriptional activation of Wnt signalling.

Malignant Peritoneal Mesothelioma in an Infant With Familial ATM Mutations.

Ataxia-telangiectasia (A-T), an autosomal recessive disorder characterized by progressive neurologic dysfunction, oculocutaneous telangiectasia, immunodeficiency, and cancer susceptibility, is caused by mutations in the ATM gene. A previous study of 4 A-T patients identified 2 rare homozygous missense mutations residing on the same allele of the ATM gene: c.1514T>C and c.1547T>C, which were shown to decrease ATM levels and increase T-cell acute lymphoblastic leukemia predisposition. We studied 5 patients from 2 consanguineous Bedouin families of the same tribe, presenting with A-T. Whole-exome sequencing data identified the 2 aforementioned mutations in ATM, which segregated within all family members as expected of autosomal recessive heredity. Interestingly, one individual was diagnosed with malignant peritoneal mesothelioma (MPM), an extremely rare neoplasm in pediatric patients. Here, we describe a case of a 4-month-old infant homozygous for the 2 ATM mutations, who developed MPM and died by the age of 2 years. To the best of our knowledge, this is the first case of peritoneal mesothelioma in an infant bearing ATM mutations, and one of the youngest pediatric mesotheliomas described. Thus, the risk of MPM might be considered in the follow-up of A-T patients, and ATM mutations sought in cases of early-onset MPM.

PAX7 mutation in a syndrome of failure to thrive, hypotonia, and global neurodevelopmental delay.

PAX7 encodes a transcription factor essential in neural crest formation, myogenesis, and pituitary lineage specification. Pax7 null mice fail to thrive and exhibit muscle weakness, dying within 3 weeks. We describe a human autosomal-recessive syndrome, with failure to thrive, severe global developmental delay, microcephaly, axial hypotonia, pyramidal signs, dystonic postures, seizures, irritability, and self-mutilation. Aside from low blood carnitine levels, biochemical and metabolic screen was normal, with growth hormone deficiency in one patient. Electromyography was normal, with no specific findings in brain MRI/MRS yet nondemonstrable neuropituitary, a finding of unclear significance. Muscle biopsy showed unaffected overall organization of muscle fibers, yet positive fetal alpha myosin staining, suggesting regeneration. Homozygosity mapping with whole-exome sequencing identified a single disease-associated mutation in PAX7, segregating as expected in the kindred with no homozygosity in 200 ethnically matched controls. Transfection experiments showed that the PAX7 splice-site mutation putatively causes nonsense-mediated mRNA decay affecting onlyPAX7 isoform 3. This isoform, expressed specifically in brain, skeletal muscle and testes, is the sole Pax7 variant normally found in mice. The human muscle phenotype is in line with that in conditional Pax7 null mutant mice, where initial aberrant histological findings resolve postnatally through muscle regeneration.

CDC174, a novel component of the exon junction complex whose mutation underlies a syndrome of hypotonia and psychomotor developmental delay.

Siblings of non-consanguineous Jewish-Ethiopian ancestry presented with congenital axial hypotonia, weakness of the abducens nerve, psychomotor developmental delay with brain ventriculomegaly, variable thinning of corpus callosum and cardiac septal defects. Homozygosity mapping identified a single disease-associated locus of 3.5 Mb on chromosome 3. Studies of a Bedouin consanguineous kindred affected with a similar recessive phenotype identified a single disease-associated 18 Mb homozygosity locus encompassing the entire 3.5 Mb locus. Whole exome sequencing demonstrated only two homozygous mutations within a shared identical haplotype of 0.6 Mb, common to both Bedouin and Ethiopian affected individuals, suggesting an ancient common founder. Only one of the mutations segregated as expected in both kindreds and was not found in Bedouin and Jewish-Ethiopian controls: c.1404A>G, p.[*468Trpext*6] in CCDC174. We showed that CCDC174 is ubiquitous, restricted to the cell nucleus and co-localized with EIF4A3. In fact, yeast-two-hybrid assay demonstrated interaction of CCDC174 with EIF4A3, a component of exon junction complex. Knockdown of the CCDC174 ortholog in Xenopus laevis embryos resulted in poor neural fold closure at the neurula stage with later embryonic lethality. Knockdown embryos exhibited a sharp reduction in expression of n-tubulin, a marker for differentiating primary neurons, and of hindbrain markers krox20 and hoxb3. The Xenopus phenotype could be rescued by the human normal, yet not the mutant CCDC174 transcripts. Moreover, overexpression of mutant but not normal CCDC174 in neuroblastoma cells caused rapid apoptosis. In line with the hypotonia phenotype, the CCDC174 mutation caused depletion of RYR1 and marked myopathic changes in skeletal muscle of affected individuals.

UNC80 mutation causes a syndrome of hypotonia, severe intellectual disability, dyskinesia and dysmorphism, similar to that caused by mutations in its interacting cation channel NALCN.

BACKGROUND: A syndrome of profound hypotonia, intellectual disability, intrauterine growth retardation with subsequent failure to thrive, dyskinesia and epilepsy was diagnosed in Bedouin Israeli families. Mild dysmorphism was evident: plagiocephaly, broad forehead with prominent nose, smooth philtrum and congenital esotropia. We set out to decipher the molecular basis of this syndrome. METHODS: Genome-wide linkage analysis and fine mapping were done. Whole exome sequencing data were filtered for candidate variants within locus. Validation and segregation of the mutation was assayed via Sanger sequencing. UNC80 expression pattern was analysed through reverse transcription PCR. RESULTS: Homozygosity mapping followed by fine mapping identified a 7.5 Mb disease-associated locus (logarithm of odds score 3.5) on chromosome 2. Whole exome and Sanger sequencing identified a single homozygous nonsense mutation within this locus, segregating within the families as expected for recessive heredity and not found in a homozygous state in 150 Bedouin controls: c.151C>T, p.(R51*) in UNC80. CONCLUSIONS: The syndrome described is caused by a mutation in UNC80, truncating most of the 3258 amino acids highly conserved encoded protein, that has no known motifs. UNC80 bridges between UNC79 and the cation channel NALCN, enabling NALCN's role in basal Na(+) leak conductance in neurons, essential for neuronal function. The phenotype caused by the UNC80 mutation resembles that previously described for homozygous NALCN mutations.

Novel GUCY2D mutation causes phenotypic variability of Leber congenital amaurosis in a large kindred.

BACKGROUND: Leber congenital amaurosis (LCA) is a severe retinal degenerative disease that manifests as blindness or poor vision in infancy. The purpose of this study was to clinically characterize and identify the cause of disease in a large inbred Bedouin Israeli tribe with LCA. METHODS: Thirty individuals of a single kindred, including eight affected with LCA, were recruited for this study. Patients' clinical data and electroretinography (ERG) findings were collected. Molecular analysis included homozygosity mapping with polymorphic markers and Sanger sequencing of candidate genes. RESULTS: Of the eight affected individuals of the kindred, nystagmus was documented in five subjects and keratoconus in three. Cataract was found in 5 of 16 eyes. Photopic and scotopic ERG performed in 5 patients were extinguished. All affected subjects were nearly blind, their visual acuity ranged between finger counting and uncertain light perception. Assuming autosomal recessive heredity of a founder mutation, studies using polymorphic markers excluded homozygosity of affected individuals at the genomic loci of all previously known genes associated with LCA, except GUCY2D. Sequencing of GUCY2D identified a novel missense mutation (c.2129C>T; p.Ala710Val) resulting in substitution of alanine by valine at position 710 within the protein kinase domain of the retina-specific enzyme guanylate cyclase 1 (GC1) encoded by GUCY2D. Molecular modeling implied that the mutation changes the conformation of the regulatory segment within the kinase styk-domain of GC1 and causes loss of its helical structure, likely inhibiting phosphorylation of threonine residue within this segment, which is needed to activate the catalytic domain of the protein. CONCLUSIONS: This is the first documentation of the p.Ala710Val mutation in GC1 and the second ever described mutation in its protein kinase domain. Our findings enlarge the scope of genetic variability of LCA, highlight the phenotypic heterogeneity found amongst individuals harboring an identical LCA mutation, and possibly provide hope for gene therapy in patients with this congenital blinding disease. As the Bedouin kindred studied originates from Saudi Arabia, the mutation found might be an ancient founder mutation in that large community.

Single cell analysis of dup15q syndrome reveals developmental and postnatal molecular changes in autism.

Duplication 15q (dup15q) syndrome is the most common genetic cause of autism spectrum disorder (ASD). Due to a higher genetic and phenotypic homogeneity compared to idiopathic autism, dup15q syndrome provides a well-defined setting to investigate ASD mechanisms. Previous bulk gene expression studies identified shared molecular changes in ASD. However, how cell type specific changes compare across different autism subtypes and how they change during development is largely unknown. In this study, we used single cell and single nucleus mRNA sequencing of dup15q cortical organoids from patient iPSCs, as well as post-mortem patient brain samples. We find cell-type specific dysregulated programs that underlie dup15q pathogenesis, which we validate by spatial resolved transcriptomics using brain tissue samples. We find degraded identity and vulnerability of deep-layer neurons in fetal stage organoids and highlight increased molecular burden of postmortem upper-layer neurons implicated in synaptic signaling, a finding shared between idiopathic ASD and dup15q syndrome. Gene co-expression network analysis of organoid and postmortem excitatory neurons uncovers modules enriched with autism risk genes. Organoid developmental modules were involved in transcription regulation via chromatin remodeling, while postmortem modules were associated with synaptic transmission and plasticity. The findings reveal a shifting landscape of ASD cellular vulnerability during brain development.

Awake prone positioning in acute hypoxaemic respiratory failure: An international expert guidance.

BACKGROUND: Awake prone positioning (APP) of non-intubated patients with acute hypoxaemic respiratory failure (AHRF) has been inconsistently adopted into routine care of patients with COVID-19, likely due to apparent conflicting evidence from recent trials. This short guideline aims to provide evidence-based recommendations for the use of APP in various clinical scenarios. METHODS: An international multidisciplinary panel, assembled for their expertise and representativeness, and supported by a methodologist, performed a systematic literature search, summarized the available evidence derived from randomized clinical trials, and developed recommendations using GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) methodology. RESULTS: The panel strongly recommends that APP rather than standard supine care be used in patients with COVID-19 receiving advanced respiratory support (high-flow nasal cannula, continuous positive airway pressure or non-invasive ventilation). Due to lack of evidence from randomized controlled trials, the panel provides no recommendation on the use of APP in patients with COVID-19 supported with conventional oxygen therapy, nor in patients with AHRF due to causes other than COVID-19. CONCLUSION: APP should be routinely implemented in patients with COVID-19 receiving advanced respiratory support.