Variants in ADD1 cause intellectual disability,corpus callosum dysgenesis,and ventriculomegaly in humans

PurposeAdducins interconnect spectrin and actin filaments to form polygonal scaffolds beneath the cell membranes and form ring-like structures in neuronal axons. Adducins regulate mouse neural development, but their function in the human brain is unknown.MethodsWe used exome sequencing to uncover ADD1 variants associated with intellectual disability (ID) and brain malformations. We studied ADD1 splice isoforms in mouse and human neocortex development with RNA sequencing, super resolution imaging, and immunoblotting. We investigated 4 variant ADD1 proteins and heterozygous ADD1 cells for protein expression and ADD1–ADD2 dimerization. We studied Add1 functions in vivo using Add1 knockout mice.ResultsWe uncovered loss-of-function ADD1 variants in 4 unrelated individuals affected by ID and/or structural brain defects. Three additional de novo copy number variations covering the ADD1 locus were associated with ID and brain malformations. ADD1 is highly expressed in the neocortex and the corpus callosum, whereas ADD1 splice isoforms are dynamically expressed between cortical progenitors and postmitotic neurons. Human variants impair ADD1 protein expression and/or dimerization with ADD2. Add1 knockout mice recapitulate corpus callosum dysgenesis and ventriculomegaly phenotypes.ConclusionOur human and mouse genetics results indicate that pathogenic ADD1 variants cause corpus callosum dysgenesis, ventriculomegaly, and/or ID.     

Mechanisms associated with Acanthamoeba castellanii (T4) phagocytosis

Using fluorescein isothiocyanate (FITC)-labelled Escherichia coli, phagocytosis in Acanthamoeba is studied. This assay is based on the quenching effect of trypan blue on FITC-labelled E. coli. Only intracellular E. coli retain their fluorescence, which are easily discriminated from non-fluorescent adherent bacteria. Acanthamoeba uptake of E. coli is significantly reduced in the presence of genistein, a protein tyrosine kinase inhibitor. In contrast, sodium orthovanadate (protein tyrosine phosphatase inhibitor) increases bacterial uptake by Acanthamoeba. Treatment of Acanthamoeba with cytochalasin D (actin polymerization inhibitor) abolished the ability of Acanthamoeba to phagocytose E. coli suggesting that tyrosine kinase-mediated signaling may play a role in Acanthamoeba phagocytosis. In addition, we showed that phosphatidylinositol 3-kinase (PI3K) plays an important role in Acanthamoeba uptake of E. coli. Role of mannose-binding protein in Acanthamoeba phagocytosis is discussed further.     

Autosomal dominant Zellweger spectrum disorder caused by de novo variants in PEX14 gene

PurposeZellweger spectrum disorders (ZSDs) are known as autosomal recessive disorders caused by defective peroxisome biogenesis due to bi-allelic pathogenic variants in any of at least 13 different PEX genes. Here, we report 2 unrelated patients who present with an autosomal dominant ZSD.MethodsWe performed biochemical and genetic studies in blood and skin fibroblasts of the patients and demonstrated the pathogenicity of the identified PEX14 variants by functional cell studies.ResultsWe identified 2 different single heterozygous de novo variants in the PEX14 genes of 2 patients diagnosed with ZSD. Both variants cause messenger RNA mis-splicing, leading to stable expression of similar C-terminally truncated PEX14 proteins. Functional studies indicated that the truncated PEX14 proteins lost their function in peroxisomal matrix protein import and cause increased degradation of peroxisomes, ie, pexophagy, thus exerting a dominant-negative effect on peroxisome functioning. Inhibition of pexophagy by different autophagy inhibitors or genetic knockdown of the peroxisomal autophagy receptor NBR1 resulted in restoration of peroxisomal functions in the patients’ fibroblasts.ConclusionOur finding of an autosomal dominant ZSD expands the genetic repertoire of ZSDs. Our study underscores that single heterozygous variants should not be ignored as possible genetic cause of diseases with an established autosomal recessive mode of inheritance.     

Lysosomal cholesterol accumulation contributes to the movement phenotypes associated with NUS1 haploinsufficiency

PurposeVariants in NUS1 are associated with a congenital disorder of glycosylation, developmental and epileptic encephalopathies, and are possible contributors to Parkinson disease pathogenesis. How the diverse functions of the NUS1-encoded Nogo B receptor (NgBR) relate to these different phenotypes is largely unknown. We present three patients with de novo heterozygous variants in NUS1 that cause a complex movement disorder, define pathogenic mechanisms in cells and zebrafish, and identify possible therapy.MethodsComprehensive functional studies were performed using patient fibroblasts, and a zebrafish model mimicking NUS1 haploinsufficiency.ResultsWe show that de novo NUS1 variants reduce NgBR and Niemann–Pick type C2 (NPC2) protein amount, impair dolichol biosynthesis, and cause lysosomal cholesterol accumulation. Reducing nus1 expression 50% in zebrafish embryos causes abnormal swim behaviors, cholesterol accumulation in the nervous system, and impaired turnover of lysosomal membrane proteins. Reduction of cholesterol buildup with 2-hydroxypropyl-ß-cyclodextrin significantly alleviates lysosomal proteolysis and motility defects.ConclusionOur results demonstrate that these NUS1 variants cause multiple lysosomal phenotypes in cells. We show that the movement deficits associated with nus1 reduction in zebrafish arise in part from defective efflux of cholesterol from lysosomes, suggesting that treatments targeting cholesterol accumulation could be therapeutic.     

Acanthamoeba keratitis in a mouse model using a novel approach

ContextAcanthamoeba is increasingly implicated in causing keratitis in patients wearing contact lens or ocular trauma and has a poor prognosis. Establishment of an animal model is critical to study the disease pathology, pathogenesis and to evaluate anti-amoebic drugs. Some studies have used contact lenses to establish Acanthamoeba keratitis (AK) in a mouse model, which is expensive and not very successful as lenses get dislodged.ObjectiveTo assess the feasibility of using parafilm (Bemis Company Inc., USA) as an alternative to contact lens for the establishment of AK in the mouse model.MethodsThirty-six Balb/c mice in three groups of six mice each for two strains of Acanthamoeba were used to induce AK. Three experimental approaches used were; i) Acanthamoeba impregnated contact lens, ii) Acanthamoeba impregnated parafilm and iii) scratching followed by inoculation of Acanthamoeba suspension. In all three models, tarsorrhaphy was performed. Infection was evaluated by clinical examination and also through microscopic examination of corneal scrapings and corneal sections.ResultsAK model was successfully established with parafilm whereas only one mouse developed AK with the use of contact lens and none with scratching and Acanthamoeba inoculation.ConclusionThe use of parafilm is convenient, reliable and cheaper and can be considered an alternative to contact lenses to induce AK in a mouse model.     

Haploinsufficiency of ARFGEF1 is associated with developmental delay,intellectual disability,and epilepsy with variable expressivity

PurposeADP ribosylation factor guanine nucleotide exchange factors (ARFGEFs) are a family of proteins implicated in cellular trafficking between the Golgi apparatus and the plasma membrane through vesicle formation. Among them is ARFGEF1/BIG1, a protein involved in axon elongation, neurite development, and polarization processes. ARFGEF1 has been previously suggested as a candidate gene for different types of epilepsies, although its implication in human disease has not been well characterized.MethodsInternational data sharing, in silico predictions, and in vitro assays with minigene study, western blot analyses, and RNA sequencing.ResultsWe identified 13 individuals with heterozygous likely pathogenic variants in ARFGEF1. These individuals displayed congruent clinical features of developmental delay, behavioral problems, abnormal findings on brain magnetic resonance image (MRI), and epilepsy for almost half of them. While nearly half of the cohort carried de novo variants, at least 40% of variants were inherited from mildly affected parents who were clinically re-evaluated by reverse phenotyping. Our in silico predictions and in vitro assays support the contention that ARFGEF1-related conditions are caused by haploinsufficiency, and are transmitted in an autosomal dominant fashion with variable expressivity.ConclusionWe provide evidence that loss-of-function variants in ARFGEF1 are implicated in sporadic and familial cases of developmental delay with or without epilepsy.     

Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder,apraxia, and insensitivity to pain

PurposeWe characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1β subunit of the cyclic AMP-dependent protein kinase A (PKA).MethodsVariants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development.ResultsRecent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs.ConclusionOur study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.     

Pathogenic variants in the paired-related homeobox 1 gene (PRRX1) cause craniosynostosis with incomplete penetrance

PurposeStudies have previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the preosteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function (LoF) variants in PRRX1 associated with craniosynostosis.MethodsTrio-based genome, exome, or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins.ResultsGenome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis, who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9 of 1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, 7 additional individuals (4 families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multisuture synostosis was present in 11 of 17 cases (65%). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis.ConclusionThis work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis.     

Biallelic DAW1 variants cause a motile ciliopathy characterized by laterality defects and subtle ciliary beating abnormalities

PurposeThe clinical spectrum of motile ciliopathies includes laterality defects, hydrocephalus, and infertility as well as primary ciliary dyskinesia when impaired mucociliary clearance results in otosinopulmonary disease. Importantly, approximately 30% of patients with primary ciliary dyskinesia lack a genetic diagnosis.MethodsClinical, genomic, biochemical, and functional studies were performed alongside in vivo modeling of DAW1 variants.ResultsIn this study, we identified biallelic DAW1 variants associated with laterality defects and respiratory symptoms compatible with motile cilia dysfunction. In early mouse embryos, we showed that Daw1 expression is limited to distal, motile ciliated cells of the node, consistent with a role in left-right patterning. daw1 mutant zebrafish exhibited reduced cilia motility and left-right patterning defects, including cardiac looping abnormalities. Importantly, these defects were rescued by wild-type, but not mutant daw1, gene expression. In addition, pathogenic DAW1 missense variants displayed reduced protein stability, whereas DAW1 loss-of-function was associated with distal type 2 outer dynein arm assembly defects involving axonemal respiratory cilia proteins, explaining the reduced cilia-induced fluid flow in particle tracking velocimetry experiments.ConclusionOur data define biallelic DAW1 variants as a cause of human motile ciliopathy and determine that the disease mechanism involves motile cilia dysfunction, explaining the ciliary beating defects observed in affected individuals.     

LST1: A multifunctional gene encoded in the MHC class III region

The LST1 gene is located in the MHC class III cluster between the MHC class I and II regions. While most genes in this cluster have been sufficiently characterised, a definitive function and expression pattern for LST1 still remains elusive. In the present review we describe its promotor, gene organisation, splice variants and expression in human tissues, cell lines and cancer. We focus on LST1 expression in inflammation and discuss known correlations with autoimmune diseases and cancer. Current data on LST1 polymorphisms and their known associations with pathologies are also discussed in detail. We summarize the potential functions that have been described for the full-length LST1 protein including its function as a transmembrane adaptor protein with inhibitory signal transduction and its role as a membrane scaffold facilitating the formation of tunnelling nanotubes. We also discuss further potential functions by compiling all known LST1-interacting proteins. Furthermore, we address knowledge gaps and conflictive issues regarding disease association, non-hematopoietic expression and the discrepancy between RNA and protein expression data.     

De novo missense variants in RAC3 cause a novel neurodevelopmental syndrome

PurposeRAC3 is an underexamined member of the Rho GTPase gene family that is expressed in the developing brain and linked to key cellular functions. De novo missense variants in the homolog RAC1 were recently associated with developmental disorders. In the RAC subfamily, transforming missense changes at certain shared residues have been observed in human cancers and previously characterized in experimental studies. The purpose of this study was to determine whether constitutional dysregulation of RAC3 is associated with human disease.MethodsWe discovered a RAC3 variant in the index case using genome sequencing, and searched for additional variants using international data-sharing initiatives. Functional effects of the variants were assessed using a multifaceted approach generalizable to most clinical laboratory settings.ResultsWe rapidly identified five individuals with de novo monoallelic missense variants in RAC3, including one recurrent change. Every participant had severe intellectual disability and brain malformations. In silico protein modeling, and prior in vivo and in situ experiments, supported a transforming effect for each of the three different RAC3 variants. All variants were observed in databases of somatic variation in cancer.ConclusionsMissense variants in RAC3 cause a novel brain disorder, likely through a mechanism of constitutive protein activation.     

Correlation between leucine rich domain and the stability of LRWD1 protein in human NT2/D1 cells

PurposeLRWD1 is a protein that contains LRR and WDs domains and is important in regulating spermatogenesis. However, the roles of LRR or WDs domains in the expression of LRWD1 remain unclear.Materials and methodsThe NT2/D1 cells separately transfected with full length of LRWD1 gene (LRWD^WT) or genes with deleted sequences in the LRR domain (LRWD1^ΔLRR), WD1 domain (LRWD1^ΔWD1), WD2 domain (LRWD1^ΔWD2), WD3 domain (LRWD1^ΔWD3) and entire three WD domains (LRWD1^Δ3×WD) were applied to investigate the expression levels of LRWD1 protein by either Western blot or flow cytometry. The associated proteins in these mutated LRWD1 proteins were identified by mass spectrometry.ResultsDeletion of the LRR domain significantly decreased the expression of LRWD1 protein. With the treatment of MG132, the LRR domain may functions in preventing LRWD1 protein from proteasome-mediated degradation. In the co-immunoprecipitation analysis, protein receptor of tumor necrosis factor 2 (TNFR2) was specifically observed to be associated with LRR-deficient LRWD1 protein.ConclusionsThe LRR domain is significantly correlated to the stability of LRWD1 protein. Determining if the stability is modulated by TNFR2 is worthy of further study.     

Autophagy protein 8 mediating autophagosome in encysting Acanthamoeba

Autophagy is an evolutionally conserved protein degradation pathway in eukaryotes. It plays essential roles during starvation, cellular differentiation, cell death, and aging by eliminating unwanted or unnecessary organelles and recycling the components for reuse. ATG8, a member of a novel ubiquitin-like protein family, is an essential component of the autophagic machinery. The present study identified and characterized autophagy protein 8 in Acanthamoeba castellanii an amphizoic amoeba causing granulomatous amoebic encephalitis and amoebic keratitis in humans. Real-time polymerase chain reaction demonstrated that the A. castellanii Atg8 (AcAtg8) gene encoding a 118 amino acid protein was highly expressed during encystation. Fluorescence microscopic analysis following transient transfection of enhanced green fluorescent protein-AcAtg8 revealed small or large vacuolar fluorescent structures in an encysting amoeba. The Atg8 fluorescent structures on the membrane were identified as autophagosomes by co-localization analysis with LysoTracker. Chemically synthesized small interfering RNA against AcAtg8 reduced the encystation efficiency and inhibited autophagosome formation in Acanthamoeba.     

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay,movement disorder,and metabolic abnormalities

PurposeThis study aimed to establish the genetic cause of a novel autosomal recessive neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.MethodsWe performed a detailed clinical characterization of 4 unrelated individuals from consanguineous families with a neurodevelopmental disorder. We used exome sequencing or targeted-exome sequencing, cosegregation, in silico protein modeling, and functional analyses of variants in HEK293 cells and Drosophila melanogaster, as well as in proband-derived fibroblast cells.ResultsIn the 4 individuals, we identified 3 novel homozygous variants in oxoglutarate dehydrogenase (OGDH) (NM_002541.3), which encodes a subunit of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase. In silico homology modeling predicts that c.566C>T:p.(Pro189Leu) and c.890C>A:p.(Ser297Tyr) variants interfere with the structure and function of OGDH. Fibroblasts from individual 1 showed that the p.(Ser297Tyr) variant led to a higher degradation rate of the OGDH protein. OGDH protein with p.(Pro189Leu) or p.(Ser297Tyr) variants in HEK293 cells showed significantly lower levels than the wild-type protein. Furthermore, we showed that expression of Drosophila Ogdh (dOgdh) carrying variants homologous to p.(Pro189Leu) or p.(Ser297Tyr), failed to rescue developmental lethality caused by loss of dOgdh. SpliceAI, a variant splice predictor, predicted that the c.935G>A:p.(Arg312Lys)/p.(Phe264_Arg312del) variant impacts splicing, which was confirmed through a mini-gene assay in HEK293 cells.ConclusionWe established that biallelic variants in OGDH cause a neurodevelopmental disorder with metabolic and movement abnormalities.     

The pleiotropy associated with de novo variants in CHD4, CNOT3, and SETD5 extends to moyamoya angiopathy

PurposeMoyamoya angiopathy (MMA) is a cerebrovascular disease characterized by occlusion of large arteries, which leads to strokes starting in childhood. Twelve altered genes predispose to MMA but the majority of cases of European descent do not have an identified genetic trigger.MethodsExome sequencing from 39 trios were analyzed.ResultsWe identified four de novo variants in three genes not previously associated with MMA: CHD4, CNOT3, and SETD5. Identification of additional rare variants in these genes in 158 unrelated MMA probands provided further support that rare pathogenic variants in CHD4 and CNOT3 predispose to MMA. Previous studies identified de novo variants in these genes in children with developmental disorders (DD), intellectual disability, and congenital heart disease.ConclusionThese genes encode proteins involved in chromatin remodeling, and taken together with previously reported genes leading to MMA-like cerebrovascular occlusive disease (YY1AP1, SMARCAL1), implicate disrupted chromatin remodeling as a molecular pathway predisposing to early onset, large artery occlusive cerebrovascular disease. Furthermore, these data expand the spectrum of phenotypic pleiotropy due to alterations of CHD4, CNOT3, and SETD5 beyond DD to later onset disease in the cerebrovascular arteries and emphasize the need to assess clinical complications into adulthood for genes associated with DD.