Structural basis of transcription regulation by CNC family transcription factor, Nrf2.

Several basic leucine zipper (bZIP) transcription factors have accessory motifs in their DNA-binding domains, such as the CNC motif of CNC family or the EHR motif of small Maf (sMaf) proteins. CNC family proteins heterodimerize with sMaf proteins to recognize CNC-sMaf binding DNA elements (CsMBEs) in competition with sMaf homodimers, but the functional role of the CNC motif remains elusive. In this study, we report the crystal structures of Nrf2/NFE2L2, a CNC family protein regulating anti-stress transcriptional responses, in a complex with MafG and CsMBE. The CNC motif restricts the conformations of crucial Arg residues in the basic region, which form extensive contact with the DNA backbone phosphates. Accordingly, the Nrf2-MafG heterodimer has approximately a 200-fold stronger affinity for CsMBE than canonical bZIP proteins, such as AP-1 proteins. The high DNA affinity of the CNC-sMaf heterodimer may allow it to compete with the sMaf homodimer on target genes without being perturbed by other low-affinity bZIP proteins with similar sequence specificity.

Amino Acid Polymorphism in Hepatitis B Virus Associated With Functional Cure.

BACKGROUND & AIMS: To provide an adequate treatment strategy for chronic hepatitis B, it is essential to know which patients are expected to have a good prognosis and which patients do not require therapeutic intervention. Previously, we identified the substitution of isoleucine to leucine at amino acid 97 (I97L) in the hepatitis B core region as a key predictor among patients with stable hepatitis. In this study, we attempted to identify the point at which I97L affects the hepatitis B virus (HBV) life cycle and to elucidate the underlying mechanisms governing the stabilization of hepatitis. METHODS: To confirm the clinical features of I97L, we used a cohort of hepatitis B e antigen-negative patients with chronic hepatitis B infected with HBV-I97 wild-type (wt) or HBV-I97L. The effects of I97L on viral characteristics were evaluated by in vitro HBV production and infection systems with the HBV reporter virus and cell culture-generated HBV. RESULTS: The ratios of reduction in hepatitis B surface antigen and HBV DNA were higher in patients with HBV-I97L than in those with HBV-I97wt. HBV-I97L exhibited lower infectivity than HBV-I97wt in both infection systems with reporter HBV and cell culture-generated HBV. HBV-I97L virions exhibiting low infectivity primarily contained a single-stranded HBV genome. The lower efficiency of cccDNA synthesis was demonstrated after infection of HBV-I97L or transfection of the molecular clone of HBV-I97L. CONCLUSIONS: The I97L substitution reduces the level of cccDNA through the generation of immature virions with single-stranded genomes. This I97L-associated low efficiency of cccDNA synthesis may be involved in the stabilization of hepatitis.

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities.

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

Anti-viral effects of interferon-λ3 on hepatitis B virus infection in cell culture.

AIM: Interferon (IFN)-λ3 is known to have antiviral effects against various pathogens. Recently, it has been reported that the production of IFN-λ3 in colon cells after the administration of nucleotide analogs is expected to reduce hepatitis B surface antigen in chronic hepatitis B patients. Here, we aimed to prove the antiviral effects of IFN-λ3 on hepatitis B virus (HBV) by using an in vitro HBV production and infection system. METHODS: We used HepG2.2.15-derived HBV as an inoculum and the replication-competent molecular clone of HBV as a replication model. RESULTS: By administering IFN-λ3 to HepG2 cells transfected with the HBV molecular clone, the production of hepatitis B surface antigen and hepatitis B core-related antigen was reduced dose-dependently. IFN-λ3 treatment also reduced the number of HBV-positive cells and the synthesis of covalently closed circular DNA after infection of HepG2.2.15-derived HBV to sodium taurocholate cotransporting polypeptide-transduced HepG2 cells. The inhibitory effect on HBV infection by IFN-λ3 was confirmed by using a recombinant a HBV reporter virus system. To elucidate the underlying mechanisms of the anti-HBV effect of IFN-λ3, we assessed the transcription of HBV RNA and the production of core-associated HBV DNA in HBV molecular clone-transfected HepG2 cells, and found that both parameters were reduced by IFN-λ3. CONCLUSIONS: We observed that the administration of IFN-λ3 inhibits HBV infection and the production of HBV proteins at the HBV RNA transcription level. This finding provides novel insight into the treatment of chronic hepatitis B patients with the administration or induction of IFN-λ3.

Hepatitis B Virus Genotype-Dependent Vulnerability of Infected Cells to Immune Reaction in the Early Phase of Infection.

Infection with hepatitis B virus (HBV) genotype (GT)-A has been reported to predispose patients to chronic infection. To explore the immune responses in infection with different HBV genotypes and clarify the genotype-dependent pathogenicity, a system mimicking the immune reaction during the early phase of HBV infection is indispensable. To this end, we established a coculture system with the replication-competent HBV molecular clone-transfected HepG2 cells and immortalized human natural killer (NK) cells, NK-92MI. Using this system, we evaluated HBV genotype dependency in NK functions and cell death of HBV positive HepG2 cells induced by NK cells or administration of tumor necrosis factor (TNF) by use of flow cytometry. After coculture with NK cells, we found that GT-A-positive HepG2 cells exhibited lower susceptibility to NK cell-induced cell death than GT-B- or GT-C-positive HepG2 cells. The NK responses of degranulation and cytokine production were not different among transfected HBV genotypes in cocultured cells. The expression levels of death receptors in HBV-transfected HepG2 cells were not different. In GT-A-positive cells, a similar low susceptibility was detected by the external administration of TNF, although relatively higher susceptibility was observed in GT-B- and GT-C-positive cells than in GT-A-positive cells. The activation of caspase signaling was revealed to be responsible for this genotype-dependent susceptibility. In conclusion, our results indicate that the HBV genotype does not influence the NK cell function itself but rather cell vulnerability through the TNF signal pathway. This observation may explain the high chronicity rate of HBV GT-A strains even in adult infections.

Ataxic phenotype with altered CaV3.1 channel property in a mouse model for spinocerebellar ataxia 42.

Spinocerebellar ataxia 42 (SCA42) is a neurodegenerative disorder recently shown to be caused by c.5144G > A (p.Arg1715His) mutation in CACNA1G, which encodes the T-type voltage-gated calcium channel CaV3.1. Here, we describe a large Japanese family with SCA42. Postmortem pathological examination revealed severe cerebellar degeneration with prominent Purkinje cell loss without ubiquitin accumulation in an SCA42 patient. To determine whether this mutation causes ataxic symptoms and neurodegeneration, we generated knock-in mice harboring c.5168G > A (p.Arg1723His) mutation in Cacna1g, corresponding to the mutation identified in the SCA42 family. Both heterozygous and homozygous mutants developed an ataxic phenotype from the age of 11-20 weeks and showed Purkinje cell loss at 50 weeks old. Degenerative change of Purkinje cells and atrophic thinning of the molecular layer were conspicuous in homozygous knock-in mice. Electrophysiological analysis of Purkinje cells using acute cerebellar slices from young mice showed that the point mutation altered the voltage dependence of CaV3.1 channel activation and reduced the rebound action potentials after hyperpolarization, although it did not significantly affect the basic properties of synaptic transmission onto Purkinje cells. Finally, we revealed that the resonance of membrane potential of neurons in the inferior olivary nucleus was decreased in knock-in mice, which indicates that p.Arg1723His CaV3.1 mutation affects climbing fiber signaling to Purkinje cells. Altogether, our study shows not only that a point mutation in CACNA1G causes an ataxic phenotype and Purkinje cell degeneration in a mouse model, but also that the electrophysiological abnormalities at an early stage of SCA42 precede Purkinje cell loss.

Germline-Derived Gain-of-Function Variants of Gsα-Coding GNAS Gene Identified in Nephrogenic Syndrome of Inappropriate Antidiuresis.

BACKGROUND: The stimulatory G-protein α-subunit encoded by GNAS exons 1-13 (GNAS-Gsα) mediates signal transduction of multiple G protein-coupled receptors, including arginine vasopressin receptor 2 (AVPR2). Various germline-derived loss-of-function GNAS-Gsα variants of maternal and paternal origin have been found in pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism, respectively. Specific somatic gain-of-function GNAS-Gsα variants have been detected in McCune-Albright syndrome and may result in phosphate wasting. However, no germline-derived gain-of-function variant has been identified, implying that such a variant causes embryonic lethality. METHODS: We performed whole-exome sequencing in two families with dominantly inherited nephrogenic syndrome of inappropriate antidiuresis (NSIAD) as a salient phenotype after excluding a gain-of-function variant of AVPR2 and functional studies for identified variants. RESULTS: Whole-exome sequencing revealed two GNAS-Gsα candidate variants for NSIAD: GNAS-Gsα p.(F68_G70del) in one family and GNAS-Gsα p.(M255V) in one family. Both variants were absent from public and in-house databases. Of genes with rare variants, GNAS-Gsα alone was involved in AVPR2 signaling and shared by the families. Protein structural analyses revealed a gain-of-function-compatible conformational property for p.M255V-Gsα, although such assessment was not possible for p.F68_G70del-Gsα. Both variants had gain-of-function effects that were significantly milder than those of McCune-Albright syndrome-specific somatic Gsα variants. Model mice for p.F68_G70del-Gsα showed normal survivability and NSIAD-compatible phenotype, whereas those for p.M255V-Gsα exhibited severe failure to thrive. CONCLUSIONS: This study shows that germline-derived gain-of-function rare variants of GNAS-Gsα exist and cause NSIAD as a novel Gsα-mediated genetic disease. It is likely that AVPR2 signaling is most sensitive to GNAS-Gsα's gain-of-function effects.

Biallelic COLGALT1 variants are associated with cerebral small vessel disease.

OBJECTIVE: Approximately 5% of cerebral small vessel diseases are hereditary, which include COL4A1/COL4A2-related disorders. COL4A1/COL4A2 encode type IV collagen α1/2 chains in the basement membranes of cerebral vessels. COL4A1/COL4A2 mutations impair the secretion of collagen to the extracellular matrix, thereby resulting in vessel fragility. The diagnostic yield for COL4A1/COL4A2 variants is around 20 to 30%, suggesting other mutated genes might be associated with this disease. This study aimed to identify novel genes that cause COL4A1/COL4A2-related disorders. METHODS: Whole exome sequencing was performed in 2 families with suspected COL4A1/COL4A2-related disorders. We validated the role of COLGALT1 variants by constructing a 3-dimensional structural model, evaluating collagen ß (1-O) galactosyltransferase 1 (ColGalT1) protein expression and ColGalT activity by Western blotting and collagen galactosyltransferase assays, and performing in vitro RNA interference and rescue experiments. RESULTS: Exome sequencing demonstrated biallelic variants in COLGALT1 encoding ColGalT1, which was involved in the post-translational modification of type IV collagen in 2 unrelated patients: c.452 T > G (p.Leu151Arg) and c.1096delG (p.Glu366Argfs*15) in Patient 1, and c.460G > C (p.Ala154Pro) and c.1129G > C (p.Gly377Arg) in Patient 2. Three-dimensional model analysis suggested that p.Leu151Arg and p.Ala154Pro destabilized protein folding, which impaired enzymatic activity. ColGalT1 protein expression and ColGalT activity in Patient 1 were undetectable. RNA interference studies demonstrated that reduced ColGalT1 altered COL4A1 secretion, and rescue experiments showed that mutant COLGALT1 insufficiently restored COL4A1 production in cells compared with wild type. INTERPRETATION: Biallelic COLGALT1 variants cause cerebral small vessel abnormalities through a common molecular pathogenesis with COL4A1/COL4A2-related disorders. Ann Neurol 2018;84:843-853.

Homozygous splicing mutation in NUP133 causes Galloway-Mowat syndrome.

OBJECTIVE: Galloway-Mowat syndrome (GAMOS) is a neural and renal disorder, characterized by microcephaly, brain anomalies, and early onset nephrotic syndrome. Biallelic mutations in WDR73 and the 4 subunit genes of the KEOPS complex are reported to cause GAMOS. Furthermore, an identical homozygous NUP107 (nucleoporin 107kDa) mutation was identified in 4 GAMOS-like families, although biallelic NUP107 mutations were originally identified in steroid-resistant nephrotic syndrome. NUP107 and NUP133 (nucleoporin 133kDa) are interacting subunits of the nuclear pore complex in the nuclear envelope during interphase, and these proteins are also involved in centrosome positioning and spindle assembly during mitosis. METHODS: Linkage analysis and whole exome sequencing were performed in a previously reported GAMOS family with brain atrophy and steroid-resistant nephrotic syndrome. RESULTS: We identified a homozygous NUP133 mutation, c.3335-11T>A, which results in the insertion of 9bp of intronic sequence between exons 25 and 26 in the mutant transcript. NUP133 and NUP107 interaction was impaired by the NUP133 mutation based on an immunoprecipitation assay. Importantly, focal cortical dysplasia type IIa was recognized in the brain of an autopsied patient and focal segmental glomerulosclerosis was confirmed in the kidneys of the 3 examined patients. A nup133-knockdown zebrafish model exhibited microcephaly, fewer neuronal cells, underdeveloped glomeruli, and fusion of the foot processes of the podocytes, which mimicked human GAMOS features. nup133 morphants could be rescued by human wild-type NUP133 mRNA but not by mutant mRNA. INTERPRETATION: These data indicate that the biallelic NUP133 loss-of-function mutation causes GAMOS. Ann Neurol 2018;84:814-828.

GRIN2D variants in three cases of developmental and epileptic encephalopathy.

N-methyl-d-aspartate (NMDA) receptors are glutamate-activated ion channels that are widely distributed in the central nervous system and essential for brain development and function. Dysfunction of NMDA receptors has been associated with various neurodevelopmental disorders. Recently, a de novo recurrent GRIN2D missense variant was found in two unrelated patients with developmental and epileptic encephalopathy. In this study, we identified by whole exome sequencing novel heterozygous GRIN2D missense variants in three unrelated patients with severe developmental delay and intractable epilepsy. All altered residues were highly conserved across vertebrates and among the four GluN2 subunits. Structural consideration indicated that all three variants are probably to impair GluN2D function, either by affecting intersubunit interaction or altering channel gating activity. We assessed the clinical features of our three cases and compared them to those of the two previously reported GRIN2D variant cases, and found that they all show similar clinical features. This study provides further evidence of GRIN2D variants being causal for epilepsy. Genetic diagnosis for GluN2-related disorders may be clinically useful when considering drug therapy targeting NMDA receptors.

Vitamin D derivatives inhibit hepatitis C virus production through the suppression of apolipoprotein.

Supplementation with vitamin D (VD) has been reported to improve the efficacy of interferon-based therapy for chronic hepatitis C. We found that 25-hydroxyvitamin D3 (25-(OH)D3), one of the metabolites of VD, has antiviral effects by inhibiting the infectious virus production of the hepatitis C virus (HCV). In this study, to clarify the underlying mechanisms of the anti-HCV effects, we searched VD derivatives that have anti-HCV effects and identified the common target molecule in the HCV life cycle by using an HCV cell culture system. After infection of Huh-7.5.1 cells with cell culture-generated HCV, VD derivatives were added to culture media, and the propagation of HCV was assessed by measuring the HCV core antigen levels in culture media and cell lysates. To determine the step in the HCV life cycle affected by these compounds, the single-cycle virus production assay was used with a CD81-negative cell line. Of the 14 structural derivatives of VD, an anti-HCV effect was detected in 9 compounds. Cell viability was not affected by these effective compounds. The 2 representative VD derivatives inhibited the infectious virus production in the single-cycle virus production assay. Treatment with these compounds and 25-(OH)D3 suppressed the expression of apolipoprotein A1 and C3, which are known to be involved in infectious virus production of HCV, and the knockdown of these apolipoproteins reduced infectious virus production. In conclusion, we identified several compounds with anti-HCV activity by screening VD derivatives. These compounds reduce the infectious virus production of HCV by suppressing the expression of apolipoproteins in host cells.

Evaluation of antiviral effects of novel NS5A inhibitors in hepatitis C virus cell culture system with full-genome infectious clones.

Nonstructural protein 5A (NS5A) inhibitors of hepatitis C virus (HCV) are known to have potent anti-viral effects; however, these inhibitors have limited activities on strains with resistant-associated substitutions or non-genotype 1 strains. To overcome these shortcomings, novel NS5A inhibitors have been developed and approved for clinical application. The aim of this study was to evaluate the anti-viral effect of novel NS5A inhibitors (derivatives of odalasvir) on HCV genotype 2 strains in a cell culture system. Chimeric JFH-1 viruses replaced with NS5A of genotypes 1 and 2 were utilized to assess the genotype-specific potencies of NS5A inhibitors. We also examined full-genome infectious clones of JFH-1, J6cc, and J8cc to confirm the effects of NS5A inhibitors on genotype 2 strains. All chimeric viruses were capable of replication at similar levels in cell culture. We examined the anti-viral effects of derivatives of the novel NS5A inhibitor and compared with the first-generation NS5A inhibitor, daclatasvir (DCV). These compounds inhibited replication of chimeric JFH-1 viruses with NS5A of genotypes 1 and 2 at low concentrations in comparison with DCV. The EC50 values of J6cc and J8cc to these compounds were more than 100-fold lower than that of DCV. By long-term culture in the presence of these compounds, we obtained highly resistant variants and identified the responsible substitutions. In conclusion, novel NS5A inhibitors displayed improved potency against HCV genotype 2 strains compared with DCV. However, the activity of these compounds was impaired by emerging resistance-associated substitutions.

A novel CYCS mutation in the α-helix of the CYCS C-terminal domain causes non-syndromic thrombocytopenia.

We report a patient with thrombocytopenia from a Japanese family with hemophilia A spanning four generations. Various etiologies of thrombocytopenia, including genetic, immunological, and hematopoietic abnormalities, determine the prognosis for this disease. In this study, we identified a novel heterozygous mutation in a gene encoding cytochrome c, somatic (CYCS, MIM123970) using whole exome sequencing. This variant (c.301_303del:p.Lys101del) is located in the α-helix of the cytochrome c (CYCS) C-terminal domain. In silico structural analysis suggested that this mutation results in protein folding instability. CYCS is one of the key factors regulating the intrinsic apoptotic pathway and the mitochondrial respiratory chain. Using the yeast model system, we clearly demonstrated that this one amino acid deletion (in-frame) resulted in significantly reduced cytochrome c protein expression and functional defects in the mitochondrial respiratory chain, indicating that the loss of function of cytochrome c underlies thrombocytopenia. The clinical features of known CYCS variants have been reported to be confined to mild or asymptomatic thrombocytopenia, as was observed for the patient in our study. This study clearly demonstrates that thrombocytopenia can result from CYCS loss-of-function variants.

Causal relationship between acute pancreatitis and methylprednisolone pulse therapy for fulminant autoimmune hepatitis: a case report and review of literature.

BACKGROUND: A causal relationship between acute pancreatitis and administration of glucocorticoids remains a matter of debate, since most of the reported cases were diagnosed with systemic vascular diseases (including systemic lupus erythematosus and polyarteritis nodosa) that may be responsible for the pancreatitis. CASE PRESENTATION: We report a case of a 51-year-old woman who developed acute pancreatitis after receiving methylprednisolone pulse therapy for the treatment of fulminant autoimmune hepatitis (AIH). She was admitted to our hospital because of overt jaundice and back pain. Since her liver dysfunction deteriorated progressively, a liver biopsy was performed and a diagnosis of AIH was established. She was given intravenous methylprednisolone pulse therapy at 1000 mg/day for 3 days, and oral prednisolone at 40 mg/day thereafter. While her liver function improved rapidly, she started complaining of mild back pain and serum amylase and lipase levels were elevated from 5 days after the initiation of steroid therapy. A CT scan revealed mildly edematous changes around the pancreas, leading to a diagnosis of acute pancreatitis. After tapering off prednisolone, back pain disappeared, and elevated serum amylase was normalized without exacerbation of AIH. A systematic literature review identified 8 cases of acute pancreatitis developing after administration of corticosteroid pulse therapy with a median latent period of 5 days. CONCLUSIONS: The present case and reports in the literature suggest that steroid pulse therapy may cause acute pancreatitis in patients having no signs of systemic vasculitis.

De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy.

V-type proton (H+) ATPase (v-ATPase) is a multi-subunit proton pump that regulates pH homeostasis in all eukaryotic cells; in neurons, v-ATPase plays additional and unique roles in synapse function. Through whole exome sequencing, we identified de novo heterozygous mutations (p.Pro27Arg, p.Asp100Tyr, p.Asp349Asn, p.Asp371Gly) in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathy with epilepsy. Early manifestations, observed in all patients, were developmental delay and febrile seizures, evolving to encephalopathy with profound delay, hypotonic/dyskinetic quadriparesis and intractable multiple seizure types in two patients (p.Pro27Arg, p.Asp100Tyr), and to moderate delay with milder epilepsy in the other two (p.Asp349Asn, p.Asp371Gly). Modelling performed on the available prokaryotic and eukaryotic structures of v-ATPase predicted p.Pro27Arg to perturb subunit interaction, p.Asp100Tyr to cause steric hindrance and destabilize protein folding, p.Asp349Asn to affect the catalytic function and p.Asp371Gly to impair the rotation process, necessary for proton transport. We addressed the impact of p.Asp349Asn and p.Asp100Tyr mutations on ATP6V1A expression and function by analysing ATP6V1A-overexpressing HEK293T cells and patients' lymphoblasts. The p.Asp100Tyr mutant was characterized by reduced expression due to increased degradation. Conversely, no decrease in expression and clearance was observed for p.Asp349Asn. In HEK293T cells overexpressing either pathogenic or control variants, p.Asp349Asn significantly increased LysoTracker® fluorescence with no effects on EEA1 and LAMP1 expression. Conversely, p.Asp100Tyr decreased both LysoTracker® fluorescence and LAMP1 levels, leaving EEA1 expression unaffected. Both mutations decreased v-ATPase recruitment to autophagosomes, with no major impact on autophagy. Experiments performed on patients' lymphoblasts using the LysoSensor™ probe revealed lower pH of endocytic organelles for p.Asp349Asn and a reduced expression of LAMP1 with no effect on the pH for p.Asp100Tyr. These data demonstrate gain of function for p.Asp349Asn characterized by an increased proton pumping in intracellular organelles, and loss of function for p.Asp100Tyr with decreased expression of ATP6V1A and reduced levels of lysosomal markers. We expressed p.Asp349Asn and p.Asp100Tyr in rat hippocampal neurons and confirmed significant and opposite effects in lysosomal labelling. However, both mutations caused a similar defect in neurite elongation accompanied by loss of excitatory inputs, revealing that altered lysosomal homeostasis markedly affects neurite development and synaptic connectivity. This study provides evidence that de novo heterozygous ATP6V1A mutations cause a developmental encephalopathy with a pathomechanism that involves perturbations of lysosomal homeostasis and neuronal connectivity, uncovering a novel role for v-ATPase in neuronal development.

De novo hotspot variants in CYFIP2 cause early-onset epileptic encephalopathy.

OBJECTIVE: The cytoplasmic fragile X mental retardation 1 interacting proteins 2 (CYFIP2) is a component of the WASP-family verprolin-homologous protein (WAVE) regulatory complex, which is involved in actin dynamics. An obvious association of CYFIP2 variants with human neurological disorders has never been reported. Here, we identified de novo hotspot CYFIP2 variants in neurodevelopmental disorders and explore the possible involvement of the CYFIP2 mutants in the WAVE signaling pathway. METHODS: We performed trio-based whole-exome sequencing (WES) in 210 families and case-only WES in 489 individuals with epileptic encephalopathies. The functional effect of CYFIP2 variants on WAVE signaling was evaluated by computational structural analysis and in vitro transfection experiments. RESULTS: We identified three de novo CYFIP2 variants at the Arg87 residue in 4 unrelated individuals with early-onset epileptic encephalopathy. Structural analysis indicated that the Arg87 residue is buried at an interface between CYFIP2 and WAVE1, and the Arg87 variant may disrupt hydrogen bonding, leading to structural instability and aberrant activation of the WAVE regulatory complex. All mutant CYFIP2 showed comparatively weaker interactions to the VCA domain than wild-type CYFIP2. Immunofluorescence revealed that ectopic speckled accumulation of actin and CYFIP2 was significantly increased in cells transfected with mutant CYFIP2. INTERPRETATION: Our findings suggest that de novo Arg87 variants in CYFIP2 have gain-of-function effects on the WAVE signaling pathway and are associated with severe neurological disorders. Ann Neurol 2018;83:794-806.

De novo variants in CAMK2A and CAMK2B cause neurodevelopmental disorders.

Objective: α (CAMK2A) and ß (CAMK2B) isoforms of Calcium/calmodulin-dependent protein kinase II (CaMKII) play a pivotal role in neuronal plasticity and in learning and memory processes in the brain. Here, we explore the possible involvement of α- and ß-CaMKII variants in neurodevelopmental disorders. Methods: Whole-exome sequencing was performed for 976 individuals with intellectual disability, developmental delay, and epilepsy. The effect of CAMK2A and CAMK2B variants on CaMKII structure and firing of neurons was evaluated by computational structural analysis, immunoblotting, and electrophysiological analysis. Results: We identified a total of five de novo CAMK2A and CAMK2B variants in three and two individuals, respectively. Seizures were common to three individuals with CAMK2A variants. Using a minigene splicing assay, we demonstrated that a splice site variant caused skipping of exon 11 leading to an in-frame deletion of the regulatory segment of CaMKII α. By structural analysis, four missense variants are predicted to impair the interaction between the kinase domain and the regulatory segment responsible for the autoinhibition of its kinase activity. The Thr286/Thr287 phosphorylation as a result of release from autoinhibition was increased in three mutants when the mutants were stably expressed in Neuro-2a neuroblastoma cells. Expression of a CaMKII α mutant in primary hippocampal neurons significantly increased A-type K+ currents, which facilitated spike repolarization of single action potentials. Interpretation: Our data highlight the importance of CaMKII α and CaMKII ß and their autoinhibitory regulation in human brain function, and suggest the enhancement of A-type K+ currents as a possible pathophysiological basis.

Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling.

The number of amino acid substitutions in the interferon (IFN) sensitivity-determining region (ISDR) of hepatitis C virus (HCV) NS5A is a strong predictor for the outcome of IFN-based treatment. To assess the involvement of ISDR in the HCV life cycle and to clarify the molecular mechanisms influencing IFN susceptibility, we used recombinant JFH-1 viruses with NS5A of the genotype 1b Con1 strain (JFH1/5ACon1) and with NS5A ISDR containing 7 amino acid substitutions (JFH1/5ACon1/i-7mut), and compared the virus propagation and the induction of interferon-stimulated genes (ISGs). By transfecting RNAs of these strains into HuH-7-derived cells, we found that the efficiency of infectious virus production of JFH1/5ACon1/i-7mut was attenuated compared with JFH1/5ACon1. After transfecting full-length HCV RNA into HepaRG cells, the mRNA expression of ISGs was sufficiently induced by IFN treatment in JFH1/5ACon1/i-7mut-transfected but not in JFH1/5ACon1-transfected cells. These data suggested that the NS5A-mediated inhibition of ISG induction was deteriorated by amino acid substitutions in the ISDR. In conclusion, using recombinant JFH-1 viruses, we demonstrated that HCV NS5A is associated with infectious virus production and the inhibition of IFN signaling, and amino acid substitutions in the NS5A ISDR deteriorate these functions. These observations explain the strain-specific evasion of IFN signaling by HCV.

A homozygous NOP14 variant is likely to cause recurrent pregnancy loss.

Recurrent pregnancy loss is newly defined as more than two consecutive miscarriages. Recurrent pregnancy loss occurs in <5% of total pregnancies. The cause in approximately 40-60% of recurrent pregnancy loss cases remains elusive and must be determined. We investigated two unrelated Iranian consanguineous families with recurrent pregnancy loss. We performed exome sequencing using DNA from a miscarriage tissue and identified a homozygous NOP14 missense variant (c.[136C>G];[136C>G]) in both families. NOP14 is an evolutionally conserved protein among eukaryotes and is required for 18S rRNA processing and 40S ribosome biogenesis. Interestingly, in zebrafish, homozygous mutation of nop14 (possibly loss of function) resulting from retrovirus-mediated insertional mutagenesis led to embryonic lethality at 5 days after fertilization, mimicking early pregnancy loss in humans. Similarly, it is known that the nop14-null yeast is inviable. These data suggest that the homozygous NOP14 mutation is likely to cause recurrent pregnancy loss. Furthermore, this study shows that exome sequencing is very useful to determine the etiology of unsolved recurrent pregnancy loss.