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Item does not contain fulltext The vast majority of human genes encode multiple isoforms through alternative splicing, and the temporal and spatial regulation of those isoforms is critical for organismal development and function. The spliceosome, which regulates and executes splicing reactions, is primarily composed of small nuclear ribonucleoproteins (snRNPs) that consist of small nuclear RNAs (snRNAs) and protein subunits. snRNA gene transcription is initiated by the snRNA-activating protein complex (SNAPc). Here, we report ten individuals, from eight families, with bi-allelic, deleterious SNAPC4 variants. SNAPC4 encoded one of the five SNAPc subunits that is critical for DNA binding. Most affected individuals presented with delayed motor development and developmental regression after the first year of life, followed by progressive spasticity that led to gait alterations, paraparesis, and oromotor dysfunction. Most individuals had cerebral, cerebellar, or basal ganglia volume loss by brain MRI. In the available cells from affected individuals, SNAPC4 abundance was decreased compared to unaffected controls, suggesting that the bi-allelic variants affect SNAPC4 accumulation. The depletion of SNAPC4 levels in HeLa cell lines via genomic editing led to decreased snRNA expression and global dysregulation of alternative splicing. Analysis of available fibroblasts from affected individuals showed decreased snRNA expression and global dysregulation of alternative splicing compared to unaffected cells. Altogether, these data suggest that these bi-allelic SNAPC4 variants result in loss of function and underlie the neuroregression and progressive spasticity in these affected individuals.

FINCA syndrome [MIM: 618278] is an autosomal recessive multisystem disorder characterized by fibrosis, neurodegeneration and cerebral angiomatosis. To date, 13 patients from nine families with biallelic NHLRC2 variants have been published. In all of them, the recurrent missense variant p.(Asp148Tyr) was detected on at least one allele. Common manifestations included lung or muscle fibrosis, respiratory distress, developmental delay, neuromuscular symptoms and seizures often followed by early death due to rapid disease progression.Here, we present 15 individuals from 12 families with an overlapping phenotype associated with nine novel NHLRC2 variants identified by exome analysis. All patients described here presented with moderate to severe global developmental delay and variable disease progression. Seizures, truncal hypotonia and movement disorders were frequently observed. Notably, we also present the first eight cases in which the recurrent p.(Asp148Tyr) variant was not detected in either homozygous or compound heterozygous state.We cloned and expressed all novel and most previously published non-truncating variants in HEK293-cells. From the results of these functional studies, we propose a potential genotype-phenotype correlation, with a greater reduction in protein expression being associated with a more severe phenotype.Taken together, our findings broaden the known phenotypic and molecular spectrum and emphasize that NHLRC2-related disease should be considered in patients presenting with intellectual disability, movement disorders, neuroregression and epilepsy with or without pulmonary involvement.

ATP1A3 encodes the α3 subunit of the sodium-potassium ATPase, one of two isoforms responsible for powering electrochemical gradients in neurons. Heterozygous pathogenic ATP1A3 variants produce several distinct neurological syndromes, yet the molecular basis for phenotypic variability is unclear. We report a novel recurrent variant, ATP1A3(NM_152296.5):c.2324C>T; p.(Pro775Leu), in nine individuals associated with the primary clinical features of progressive or non-progressive spasticity and developmental delay/intellectual disability. No patients fulfil diagnostic criteria for ATP1A3-associated syndromes, including alternating hemiplegia of childhood, rapid-onset dystonia-parkinsonism or cerebellar ataxia-areflexia-pes cavus-optic atrophy-sensorineural hearing loss (CAPOS), and none were suspected of having an ATP1A3-related disorder. Uniquely among known ATP1A3 variants, P775L causes leakage of sodium ions and protons into the cell, associated with impaired sodium binding/occlusion kinetics favouring states with fewer bound ions. These phenotypic and electrophysiologic studies demonstrate that ATP1A3:c.2324C>T; p.(Pro775Leu) results in mild ATP1A3-related phenotypes resembling complex hereditary spastic paraplegia or idiopathic spastic cerebral palsy. Cation leak provides a molecular explanation for this genotype-phenotype correlation, adding another mechanism to further explain phenotypic variability and highlighting the importance of biophysical properties beyond ion transport rate in ion transport diseases.

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.

Neurodevelopmental disorders (NDD5) are clinically and genetically heterogenous; many such disorders are secondary to perturbation in brain development and/or function. The prevalence of NDD5 is > 3%, resulting in significant sociocultural and economic challenges to society. With recent advances in family-based genomics, rare-variant analyses, and further exploration of the Clan Genomics hypothesis, there has been a logarithmic explosion in neurogenetic "disease-associated genes" molecular etiology and biology of NDD5; however, the majority of NDD5 remain molecularly undiagnosed. We applied genome-wide screening technologies, including exome sequencing (ES) and whole-genome sequencing (WGS), to identify the molecular etiology of 234 newly enrolled subjects and 20 previously unsolved Turkish NDD families. In 176 of the 234 studied families (75.2%), a plausible and genetically parsimonious molecular etiology was identified. Out of 176 solved families, deleterious variants were identified in 218 distinct genes, further documenting the enormous genetic heterogeneity and diverse perturbations in human biology underlying NDD5. We propose 86 candidate disease-trait-associated genes for an NDD phenotype. Importantly, on the basis of objective and internally established variant prioritization criteria, we identified 51 families (51/176 = 28.9%) with multilocus pathogenic variation (MPV), mostly driven by runs of homozygosity (ROH5) - reflecting genomic segments/haplotypes that are identical-by-descent. Furthermore, with the use of additional bioinformatic tools and expansion of ES to additional family members, we established a molecular diagnosis in 5 out of 20 families (25%) who remained undiagnosed in our previously studied NDD cohort emanating from Turkey. United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Human Genome Research Institute (NHGRI) ; United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Heart Lung & Blood Institute (NHLBI) ; International Rett Syndrome Foundation (IRSF

Exome sequencing (ES) has revolutionized rare disease management, yet only ~25%–30% of patients receive a molecular diagnosis. A limiting factor is the quality of available phenotypic data. Here, we describe how deep clinicopathological phenotyping yielded a molecular diagnosis for a 19‐year‐old proband with muscular dystrophy and negative clinical ES. Deep phenotypic analysis identified two critical data points: (1) the absence of emerin protein in muscle biopsy and (2) clinical features consistent with Emery‐Dreifuss muscular dystrophy. Sequencing data analysis uncovered an ultra‐rare, intronic variant in EMD, the gene encoding emerin. The variant, NM_000117.3: c.188‐6A > G, is predicted to impact splicing by in silico tools. This case thus illustrates how better integration of clinicopathologic data into ES analysis can enhance diagnostic yield with implications for clinical practice.

Purpose: Dominant variants in the retinoic acid receptor beta (RARB) gene underlie a syndromic form of microphthalmia, known as MCOPS12, which is associated with other birth anomalies and global developmental delay with spasticity and/or dystonia. Here, we report 25 affected individuals with 17 novel pathogenic or likely pathogenic variants in RARB. This study aims to characterize the functional impact of these variants and describe the clinical spectrum of MCOPS12. Methods: We used in vitro transcriptional assays and in silico structural analysis to assess the functional relevance of RARB variants in affecting the normal response to retinoids. Results: We found that all RARB variants tested in our assays exhibited either a gain-of-function or a loss-of-function activity. Loss-of-function variants disrupted RARB function through a dominant-negative effect, possibly by disrupting ligand binding and/or coactivators’ recruitment. By reviewing clinical data from 52 affected individuals, we found that disruption of RARB is associated with a more variable phenotype than initially suspected, with the absence in some individuals of cardinal features of MCOPS12, such as developmental eye anomaly or motor impairment. Conclusion: Our study indicates that pathogenic variants in RARB are functionally heterogeneous and associated with extensive clinical heterogeneity.

The leucine-rich glioma-inactivated (LGI) family consists of four highly conserved paralogous genes, LGI1-4, that are highly expressed in mammalian central and/or peripheral nervous systems. LGI1 antibodies are detected in subjects with autoimmune limbic encephalitis and peripheral nerve hyperexcitability syndromes (PNHSs) such as Isaacs and Morvan syndromes. Pathogenic variations of LGI1 and LGI4 are associated with neurological disorders as disease traits including familial temporal lobe epilepsy and neurogenic arthrogryposis multiplex congenita 1 with myelin defects, respectively. No human disease has been reported associated with either LGI2 or LGI3. We implemented exome sequencing and family-based genomics to identify individuals with deleterious variants in LGI3 and utilized GeneMatcher to connect practitioners and researchers worldwide to investigate the clinical and electrophysiological phenotype in affected subjects. We also generated Lgi3-null mice and performed peripheral nerve dissection and immunohistochemistry to examine the juxtaparanode LGI3 microarchitecture. As a result, we identified 16 individuals from eight unrelated families with loss-of-function (LoF) bi-allelic variants in LGI3. Deep phenotypic characterization showed LGI3 LoF causes a potentially clinically recognizable PNHS trait characterized by global developmental delay, intellectual disability, distal deformities with diminished reflexes, visible facial myokymia, and distinctive electromyographic features suggestive of motor nerve instability. Lgi3-null mice showed reduced and mis-localized Kv1 channel complexes in myelinated peripheral axons. Our data demonstrate bi-allelic LoF variants in LGI3 cause a clinically distinguishable disease trait of PNHS, most likely caused by disturbed Kv1 channel distribution in the absence of LGI3.

Background The diagnosis of uncommon pediatric neuromuscular disease (NMD) is challenging due to genetic and phenotypic heterogeneity, yet is important to guide treatment, prognosis, and recurrence risk. Patients with diagnostically challenging presentations typically undergo extensive testing with variable molecular diagnostic yield. Given the advancement in next generation sequencing (NGS), we investigated the value of clinical whole exome sequencing (ES) in uncommon pediatric NMD. Methods A retrospective cohort study of 106 pediatric NMD patients with a combination of ES, chromosomal microarray (CMA), and candidate gene testing was completed at a large tertiary referral center. Results A molecular diagnosis was achieved in 37/79 (46%) patients with ES, 4/44 (9%) patients with CMA, and 15/74 (20%) patients with candidate gene testing. In 2/79 (3%) patients, a dual molecular diagnosis explaining the neuromuscular disease process was identified. A total of 42 patients (53%) who received ES remained without a molecular diagnosis at the conclusion of the study. Conclusions Due to NGS, molecular diagnostic yield of rare neurological diseases is at an all-time high. We show that ES has a higher diagnostic rate compared to other genetic tests in a complex pediatric neuromuscular disease cohort and should be considered early in the diagnostic journey for select NMD patients with challenging presentations in which a clinical diagnosis is not evident.

Negative regulator of reactive oxygen species (NRROS) is a leucine-rich repeat-containing protein that uniquely associates with latent transforming growth factor beta-1 (TGF- β1) and anchors it on the cell surface; this anchoring is required for activation of TGF-β1 in macrophages and microglia. We report six individuals from four families with bi-allelic variants in NRROS. All affected individuals had neurodegenerative disease with refractory epilepsy, developmental regression, and reduced white matter volume with delayed myelination. The clinical course in affected individuals began with normal development or mild developmental delay, and the onset of seizures occurred within the first year of life, followed by developmental regression. Intracranial calcification was detected in three individuals. The phenotypic features in affected individuals are consistent with those observed in the Nrros knockout mouse, and they overlap with those seen in the human condition associated with TGF-β1 deficiency. The disease-causing NRROS variants involve two significant functional NRROS domains. These variants result in aberrant NRROS proteins with impaired ability to anchor latent TGF-β1 on the cell surface. Using confocal microscopy in HEK293T cells, we demonstrate that wild-type and mutant NRROS proteins co-localize with latent TGF-β1 intracellularly. However, using flow cytometry, we show that our mutant NRROS proteins fail to anchor latent TGF-β1 at the cell surface in comparison to wild-type NRROS. Moreover, wild-type NRROS rescues the defect of our disease-associated mutants in presenting latent TGF-β1 to the cell surface. Taken together, our findings suggest that loss of NRROS function causes a severe childhood-onset neurodegenerative condition with features suggestive of a disordered response to inflammation.

Hereditary sensory and autonomic neuropathy type 2B (HSAN2B) is a rare autosomal recessive peripheral neuropathy caused by biallelic variants in RETREG1 (formerly FAM134B). HSAN2B is characterized by sensory impairment resulting in skin ulcerations, amputations, and osteomyelitis as well as variable weakness, spasticity, and autonomic dysfunction. Here, we report four affected individuals with recurrent osteomyelitis, ulceration, and amputation of hands and feet, sensory neuropathy, hyperhidrosis, urinary incontinence, and renal failure from a family without any known shared parental ancestry. Due to the history of chronic recurrent multifocal osteomyelitis and microcytic anemia, a diagnosis of Majeed syndrome was considered; however, sequencing of LPIN2 was negative. Family-based exome sequencing (ES) revealed a novel homozygous ultrarare RETREG1 variant NM_001034850.2:c.321G>A;p.Trp107Ter. Electrophysiological studies of the proband demonstrated axonal sensorimotor neuropathy predominantly in the lower extremities. Consistent with the lack of shared ancestry, the coefficient of inbreeding calculated from ES data was low (F = 0.002), but absence of heterozygosity (AOH) analysis demonstrated a 7.2 Mb AOH block surrounding the variant consistent with a founder allele. Two of the four affected individuals had unexplained renal failure which has not been reported in HSAN2B cases to date. Therefore, this report describes a novel RETREG1 founder allele and suggests renal failure may be an unrecognized feature of the RETREG1-disease spectrum.

© 2022 American College of Medical Genetics and GenomicsPurpose: Pathogenic variants in genes encoding ubiquitin E3 ligases are known to cause neurodevelopmental syndromes. Additional neurodevelopmental disorders associated with the other genes encoding E3 ligases are yet to be identified. Methods: Chromosomal analysis and exome sequencing were used to identify the genetic causes in 10 patients from 7 unrelated families with syndromic neurodevelopmental, seizure, and movement disorders and neurobehavioral phenotypes. Results: In total, 4 patients were found to have 3 different homozygous loss-of-function (LoF) variants, and 3 patients had 4 compound heterozygous missense variants in the candidate E3 ligase gene, HECTD4, that were rare, absent from controls as homozygous, and predicted to be deleterious in silico. In 3 patients from 2 families with Angelman-like syndrome, paralog-directed candidate gene approach detected 2 LoF variants in the other candidate E3 ligase gene, UBE3C, a paralog of the Angelman syndrome E3 ligase gene, UBE3A. The RNA studies in 4 patients with LoF variants in HECTD4 and UBE3C provided evidence for the LoF effect. Conclusion: HECTD4 and UBE3C are novel biallelic rare disease genes, expand the association of the other HECT E3 ligase group with neurodevelopmental syndromes, and could explain some of the missing heritability in patients with a suggestive clinical diagnosis of Angelman syndrome.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1–7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10–12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10–12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

Genomic sequencing and clinical genomics have demonstrated that substantial subsets of atypical and/or severe disease presentations result from multilocus pathogenic variation (MPV) causing blended phenotypes. In an infant with a severe neurodevelopmental disorder, four distinct molecular diagnoses were found by exome sequencing (ES). The blended phenotype that includes brain malformation, dysmorphism, and hypotonia was dissected using the Human Phenotype Ontology (HPO). ES revealed variants in CAPN3 (c.259C > G:p.L87V), MUSK (c.1781C > T:p.A594V), NAV2 (c.1996G > A:p.G666R), and ZC4H2 (c.595A > C:p.N199H). CAPN3, MUSK, and ZC4H2 are established disease genes linked to limb-girdle muscular dystrophy (OMIM# 253600), congenital myasthenia (OMIM# 616325), and Wieacker-Wolff syndrome (WWS; OMIM# 314580), respectively. NAV2 is a retinoic-acid responsive novel disease gene candidate with biological roles in neurite outgrowth and cerebellar dysgenesis in mouse models. Using semantic similarity, we show that no gene identified by ES individually explains the proband phenotype, but rather the totality of the clinically observed disease is explained by the combination of disease-contributing effects of the identified genes. These data reveal that multilocus pathogenic variation can result in a blended phenotype with each gene affecting a different part of the nervous system and nervous system-muscle connection. We provide evidence from this n = 1 study that in patients with MPV and complex blended phenotypes resulting from multiple molecular diagnoses, quantitative HPO analysis can allow for dissection of phenotypic contribution of both established disease genes and novel disease gene candidates not yet proven to cause human disease.

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with a novel X-linked condition characterized by diaphragm defects, variable anterior body wall anomalies, and/or facial dysmorphism. Using linkage analysis and whole exome or whole genome sequencing, we identified novel missense variants in the actin binding domains of plastin 3 (PLS3), a gene encoding an actin bundling protein, that co-segregate with disease in all families. Loss-of-function variants in PLS3 have been described previously in association with X-linked osteoporosis. To address these seemingly disparate clinical phenotypes, we performed in silico protein modeling and cellular overexpression experiments, which suggest that the affected residues in individuals with CDH are important for actin binding and result in disorganization of the actin cytoskeleton and a reduction in normal actin stress fiber formation. A mouse knock-in model of a variant identified in one of the families, p.W499C, shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal wall defects. Both the mouse model and one surviving adult patient with a PLS3 variant were observed to have increased, rather than decreased, bone mineral density. Together, these clinical and functional data in human and mouse reveal that specific missense variants affecting the actin binding domains of PLS3 may have a gain-of-function effect and cause a new Mendelian disorder.

The RNA exosome is a multi-subunit complex involved in the processing, degradation, and regulated turnover of RNA. Several subunits are linked to Mendelian disorders, including pontocerebellar hypoplasia (EXOSC3, MIM #614678; EXOSC8, MIM #616081: and EXOSC9, MIM #618065) and short stature, hearing loss, retinitis pigmentosa, and distinctive facies (EXOSC2, MIM #617763). More recently, EXOSC5 (MIM *606492) was found to underlie an autosomal recessive neurodevelopmental disorder characterized by developmental delay, hypotonia, cerebellar abnormalities, and dysmorphic facies. An unusual feature of EXOSC5-related disease is the occurrence of complete heart block requiring a pacemaker in a subset of affected individuals. Here, we provide a detailed clinical and molecular characterization of two siblings with microcephaly, developmental delay, cerebellar volume loss, hypomyelination, with cardiac conduction and rhythm abnormalities including sinus node dysfunction, intraventricular conduction delay, atrioventricular block, and ventricular tachycardia (VT) due to compound heterozygous variants in EXOSC5: (1) NM_020158.4:c.341C > T (p.Thr114Ile; pathogenic, previously reported) and (2) NM_020158.4:c.302C > A (p.Thr101Lys; novel variant). A review of the literature revealed an additional family with biallelic EXOSC5 variants and cardiac conduction abnormalities. These clinical and molecular data provide compelling evidence that cardiac conduction abnormalities and arrhythmias are part of the EXOSC5-related disease spectrum and argue for proactive screening due to potential risk of sudden cardiac death.

The Mediator multiprotein complex functions as a regulator of RNA polymerase II-catalyzed gene transcription. In this study, exome sequencing (ES) detected biallelic putative disease-causing variants in MED27, encoding Mediator Complex Subunit 27, in sixteen patients from eleven families with a novel neurodevelopmental syndrome. Patient phenotypes are highly homogeneous including global developmental delay, intellectual disability, axial hypotonia with distal spasticity, dystonic movements, and cerebellar hypoplasia. Seizures and cataracts were noted in severely affected individuals. Identification of multiple patients with biallelic MED27 variants supports the critical role of MED27 in normal human neural development, particularly for the cerebellum. This article is protected by copyright. All rights reserved.

Objective Pathogenic variants in TNNT3, the gene encoding fast skeletal muscle troponin T, were first described in autosomal dominant distal arthrogryposis type 2B2. Recently, a homozygous splice site variant, c.681+1G>A, was identified in a patient with nemaline myopathy and distal arthrogryposis. Here, we describe the second individual with congenital myopathy associated with biallelic TNNT3 variants. Methods Clinical exome sequencing data from a patient with molecularly undiagnosed congenital myopathy underwent research reanalysis. Clinical and histopathologic data were collected and compared with the single reported patient with TNNT3-related congenital myopathy. Results A homozygous TNNT3 variant, c.481-1G>A, was identified. This variant alters a consensus splice acceptor and is predicted to affect splicing by multiple in silico prediction tools. Both the patient reported here and the previously published patient exhibited limb, bulbar, and respiratory muscle weakness from birth, which improved over time. Other shared features include history of polyhydramnios, hypotonia, scoliosis, and high-arched palate. Distal arthrogryposis and nemaline rods, findings reported in the first patient with TNNT3-related congenital myopathy, were not observed in the patient reported here. Conclusions This report provides further evidence for the association of biallelic TNNT3 variants with severe recessive congenital myopathy with or without nemaline rods and distal arthrogryposis. TNNT3 sequencing and copy number analysis should be incorporated into the workup of congenital myopathies.

Listeria monocytogenes is an intracellular Gram-positive bacterium that induces expression of type I IFNs (IFN-α/IFN-β) during infection. These cytokines are detrimental to the host during infection by priming leukocytes to undergo L. monocytogenes–mediated apoptosis. Our previous studies showed that C5aR1−/− and C3aR−/− mice are highly susceptible to L. monocytogenes infection as a result of increased IFN-β–mediated apoptosis of major leukocyte cell populations, including CD4+ and CD8+ T cells. However, the mechanisms by which C3a and C5a modulate IFN-β expression during L. monocytogenes infection were not examined in these initial investigations. Accordingly, we report in this article that C5a and C3a suppress IFN-β production in response to L. monocytogenes via cyclic di-AMP (c-di-AMP), a secondary messenger molecule of L. monocytogenes, in J774A.1 macrophage-like cells and in bone marrow–derived dendritic cells (BMDCs). Moreover, C5a and C3a suppress IFN-β production by acting through their respective receptors, because no inhibition was seen in C5aR1−/− or C3aR−/− BMDCs, respectively. C5a and C3a suppress IFN-β production in a manner that is dependent on Bruton’s tyrosine kinase, p38 MAPK, and TANK-binding kinase 1 (TBK1), as demonstrated by the individual use of Bruton’s tyrosine kinase, p38 MAPK, and TBK1 inhibitors. Pretreatment of cells with C5a and C3a reduced the expression of the IFN-β signaling molecules DDX41, STING, phosphorylated TBK1, and phosphorylated p38 MAPK in wild-type BMDCs following treatment with c-di-AMP. Collectively, these data demonstrate that C3a and C5a, via direct signaling through their specific receptors, suppress IFN-β expression by modulation of a distinct innate cytosolic surveillance pathway involving DDX41, STING, and other downstream molecular targets of L. monocytogenes–generated c-di-AMP.

Pathogenic variants in ATP1A2, a gene encoding the α subunit of the Na,K-ATPase, cause familial hemiplegic migraine type 2 (FHM2). In contrast, pathogenic variants in ATP1A3, an ATP1A2 paralog, cause alternating hemiplegia of childhood (AHC), a severe neurodevelopmental disorder with infantile onset hemiplegic attacks, seizures, dystonia, chorea and developmental delay. Despite high sequence homology with ATP1A3, ATP1A2 variants rarely associate with severe phenotypes resembling those linked to ATP1A3. Here we describe two unrelated patients with infantile onset hemiplegic attacks, refractory epilepsy, movement disorders, abnormal eye movements and truncal ataxia with a shared de novo variant in ATP1A2, c.2438T > A (p.Met813Lys). The variant is not found in population databases, is predicted to be damaging by in silico analysis, and affects a highly conserved residue. Both patients experienced severe attacks with unilateral cerebral edema followed by sustained, stepwise regression. This report highlights the need to sequence ATP1A2 in the workup of patients with features of AHC that do not fulfill AHC diagnostic criteria.

EIF2AK1andEIF2AK2encode members of the Eukaryotic Translation Initiation Factor 2 Alpha Kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants inEIF2AK1andEIF2AK2have not been reported. Here, we describe the identification of eight unrelated individuals with heterozygousde novomissense variants inEIF2AK1(1/8) orEIF2AK2(7/8). Features seen in these eight individuals include white matter alterations (8/8), developmental delay (8/8), impaired language (8/8), cognitive impairment (7/8), ataxia (6/8), dysarthria in probands with verbal ability (6/6), hypotonia (6/8), hypertonia (5/8), and involuntary movements (3/8). Individuals withEIF2AK2variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and patient-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate Eukaryotic Translation Initiation Factor 2 Subunit 1, (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter disease (CACH/VWM), a leukoencephalopathy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate thatEIF2AK2missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

ObjectivePathogenic variants in TNNT3, the gene encoding fast skeletal muscle troponin T, were first described in autosomal dominant distal arthrogryposis type 2B2. Recently, a homozygous splice site variant, c.681+1G>A, was identified in a patient with nemaline myopathy and distal arthrogryposis. Here, we describe the second individual with congenital myopathy associated with biallelic TNNT3 variants.MethodsClinical exome sequencing data from a patient with molecularly undiagnosed congenital myopathy underwent research reanalysis. Clinical and histopathologic data were collected and compared with the single reported patient with TNNT3-related congenital myopathy.ResultsA homozygous TNNT3 variant, c.481-1G>A, was identified. This variant alters a consensus splice acceptor and is predicted to affect splicing by multiple in silico prediction tools. Both the patient reported here and the previously published patient exhibited limb, bulbar, and respiratory muscle weakness from birth, which improved over time. Other shared features include history of polyhydramnios, hypotonia, scoliosis, and high-arched palate. Distal arthrogryposis and nemaline rods, findings reported in the first patient with TNNT3-related congenital myopathy, were not observed in the patient reported here.ConclusionsThis report provides further evidence for the association of biallelic TNNT3 variants with severe recessive congenital myopathy with or without nemaline rods and distal arthrogryposis. TNNT3 sequencing and copy number analysis should be incorporated into the workup of congenital myopathies.

ObjectiveTo demonstrate that de novo missense single nucleotide variants (SNVs) in EIF2AK2 cause a neurodevelopmental disorder with leukoencephalopathy resembling Pelizaeus-Merzbacher disease (PMD).MethodsA retrospective chart review was performed of 2 unrelated males evaluated at a single institution with de novo EIF2AK2 SNVs identified by clinical exome sequencing (ES). Clinical and radiographic data were reviewed and summarized.ResultsBoth individuals presented in the first year of life with concern for seizures and developmental delay. Common clinical findings included horizontal and/or pendular nystagmus during infancy, axial hypotonia, appendicular hypertonia, spasticity, and episodic neurologic regression with febrile viral illnesses. MRI of the brain demonstrated severely delayed myelination in infancy. A hypomyelinating pattern was confirmed on serial imaging at age 4 years for proband 1. In proband 2, repeat imaging at age 13 months confirmed persistent delayed myelination. These clinical and radiographic features led to a strong suspicion of PMD. However, neither PLP1 copy number variants nor pathogenic SNVs were detected by chromosomal microarray and trio ES, respectively. Reanalysis of trio ES identified heterozygous de novo EIF2AK2 missense variant c.290C>T (p.Ser97Phe) in proband 1 and c.326C>T (p.Ala109Val) in proband 2.ConclusionsThe autosomal dominant EIF2AK2-related leukoencephalopathy, developmental delay, and episodic neurologic regression syndrome should be considered in the differential diagnosis for PMD and other hypomyelinating leukodystrophies (HLDs). A characteristic history of developmental regression with febrile illnesses may help distinguish it from other HLDs.

Malignant atrophic papulosis (Degos disease) is an unusual thrombotic microangiopathy of uncertain etiology. The disease characteristically involves the skin and internal organs, with nervous system involvement more common in children. We present a case with diverse neurological manifestations including cranial nerve palsies, gait instability, and urinary incontinence. The patient also developed white papular lesions on her lower extremities and back. Magnetic resonance imaging (MRI) demonstrated progressive intracranial and spinal abnormalities. Despite treatment with numerous biologic agents, the patient had persistent clinical deterioration and expired one month after admission. We highlight the extensive neurologic manifestations of Degos disease correlated with neuroradiological imaging and pathological features. Nervous system involvement in Degos disease requires careful neurologic and dermatologic exam with central nervous system (CNS) magnetic resonance imaging to distinguish it from non-organic etiologies of similar symptoms.

Listeria monocytogenes is a major cause of mortality resulting from food poisoning in the United States. In mice, C5 has been genetically linked to host resistance to listeriosis. Despite this genetic association, it remains poorly understood how C5 and its activation products, C5a and C5b, confer host protection to this Gram-positive intracellular bacterium. In this article, we show in a systemic infection model that the major receptor for C5a, C5aR1, is required for a normal robust host immune response against L. monocytogenes. In comparison with wild-type mice, C5aR1−/− mice had reduced survival and increased bacterial burden in their livers and spleens. Infected C5aR1−/− mice exhibited a dramatic reduction in all major subsets of splenocytes, which was associated with elevated caspase-3 activity and increased TUNEL staining. Because type 1 IFN has been reported to impede the host response to L. monocytogenes through the promotion of splenocyte death, we examined the effect of C5aR1 on type 1 IFN expression in vivo. Indeed, serum levels of IFN-α and IFN-β were significantly elevated in L. monocytogenes–infected C5aR1−/− mice. Similarly, the expression of TRAIL, a type 1 IFN target gene and a proapoptotic factor, was elevated in NK cells isolated from infected C5aR1−/− mice. Treatment of C5aR1−/− mice with a type 1 IFNR blocking Ab resulted in near-complete rescue of L. monocytogenes–induced mortality. Thus, these findings reveal a critical role for C5aR1 in host defense against L. monocytogenes through the suppression of type 1 IFN expression.

Listeria monocytogenes (Lm) is an intracellular Gram-positive bacterium that induces expression of type I interferons (IFN-α/IFN-β) during infection. These cytokines are detrimental to the host during infection by priming leukocytes to undergo Lm-mediated apoptosis. Our previous studies showed that C5aR1−/− and C3aR−/− mice are highly susceptible to Lm infection due to elevated IFN-β–mediated apoptosis of major leukocyte cell populations, including CD4+ and CD8+ T-cells. However, the mechanisms by which C3a and C5a modulate IFN-β expression during Lm infection were not examined in these initial investigations. Accordingly, we report here that C5a and C3a suppress IFN-β production in response to Lm via c-di-AMP, a secondary messenger molecule of Lm, in both J774A.1 macrophage-like cells and in bone marrow-derived dendritic cells (BMDCs). Moreover, C5a and C3a suppress IFN-β production by acting through their respective receptors, as no inhibition was seen in C5aR1−/− or C3aR−/− BMDCs, respectively. C5a and C3a suppress IFN-β production in a manner that is dependent on Bruton’s Tyrosine Kinase (BTK), p38 MAPK, and TANK-Binding Kinase 1 (TBK1), as demonstrated by the individual use of BTK, p38 MAPK, and TBK1 inhibitors. Pre-treatment of cells with C5a and C3a reduced the expression of the IFN-β signaling molecules DDX41, STING, phosphorylated TBK1, and phosphorylated p38 MAPK in WT BMDCs following treatment with c-di-AMP. Collectively, these data demonstrate that C3a and C5a, via direct signaling through their specific receptors, suppress IFN-β expression by modulation of a distinct innate cytosolic surveillance pathway involving DDX41, STING, and other downstream molecular targets of Lm-generated c-di-AMP.

Embryonic stem (ES) cells derived from preimplantation blastocysts and induced pluripotent stem (iPS) cells generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide a possible unlimited source of cells that could be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. Because of inherent difficulties in deriving endodermal cells from undifferentiated cell cultures, applications using lung epithelial cells derived from ES and iPS cells have lagged behind similar efforts devoted to other tissues, such as the heart and spinal cord. However, during the past several years, significant advances in culture, differentiation, and purification protocols, as well as in bioengineering methodologies, have fueled enthusiasm for the development of stem cell–based lung therapeutics. This article provides an overview of recent research achievements and discusses future technical challenges that must be met before the promise of stem cell applications for lung disease can be realized.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.