Defective monocyte plasticity and altered cAMP pathway characterize USB1-mutated poikiloderma with neutropenia Clericuzio type.

Poikiloderma with neutropenia (PN) Clericuzio type (OMIM #604173) is a rare disease with areas of skin hyper- and hypopigmentation caused by biallelic USB1 variants. The current study was spurred by poor healing of a perianal tear wound in one affected child homozygous for c.266-1G>A (p.E90Sfster8) mutation, from a family reported previously. Treatment with G-CSF/CSF3 or GM-CSF/CSF2 transiently increased neutrophil/monocytes count with no effect on wound healing. Analysis of peripheral blood revealed a lack of non-classical (CD14+/- CD16+ ) monocytes, associated with a systemic inflammatory cytokine profile, in the two affected brothers. Importantly, despite normal expression of cognate receptors, monocytes from PN patients did not respond to M-CSF or IL-34 in vitro, as determined by cytokine secretion or CD16 expression. RNAseq of monocytes showed 293 differentially expressed genes, including significant downregulation of GATA2, AKAP6 and PDE4DIP that are associated with leucocyte differentiation and cyclic adenosine monophosphate (cAMP) signalling. Notably, the plasma cAMP was significantly low in the PN patients. Our study revealed a novel association of PN with a lack of non-classical monocyte population. The defects in monocyte plasticity may contribute to disease manifestations in PN and a defective cAMP signalling may be the primary effect of the splicing errors caused by USB1 mutation.

Distinctive phenotypes in two children with novel germline RUNX1 mutations - one with myeloid malignancy and increased fetal hemoglobin.

RUNX1 associated familial platelet disorder (FPD) is a rare autosomal dominant hematologic disorder characterized by thrombocytopenia and/or altered platelet function. There is an increased propensity to develop myeloid malignancy (MM) - acute myeloid leukemia, myeloproliferative neoplasms or myelodysplastic syndrome often in association with secondary somatic variants in other genes. To date, 23 FPD-MM pediatric cases have been reported worldwide. Here, we present two new kindreds with novel RUNX1 pathogenic variants in which children are probands. The first family is a daughter/mother diad, sharing a heterozygous frameshift variant in RUNX1 gene (c.501delT p.Ser167Argfs*9). The daughter, age 13 years, presented with features resembling juvenile myelomonocytic leukemia - severe anemia, thrombocytopenia, high white cell count with blast cells, monocytosis, increased nucleated red cells and had somatic mutations with high allele burden in CUX1, PHF6, and SH2B3 genes. She also had increased fetal hemoglobin and increased LIN28B expression. The mother, who had a long history of hypoplastic anemia, had different somatic mutations- a non-coding mutation in CUX1 but none in PHF6 or SH2B3. Her fetal hemoglobin and LIN28B expression were normal. In the second kindred, the proband, now 4 years old with thrombocytopenia alone, was investigated at 3 months of age for persistent neonatal thrombocytopenia with large platelets. Molecular testing identified a heterozygous intragenic deletion in RUNX1 encompassing exon 5. His father is known to have increased bruising for several years but is unavailable for testing. These two cases illustrate the significance of secondary mutations in the development and progression of RUNX1-FPD to MM.

CD14/16 monocyte profiling in juvenile myelomonocytic leukemia.

Monocyte subset analysis by flow cytometry has been shown to be a useful diagnostic tool in chronic myelomonocytic leukemia in adults. An increase in the classical monocyte fraction (CD14++/CD16-) greater than 94.0% of total monocytes is considered highly sensitive and specific in distinguishing chronic myelomonocytic leukemia from other myeloproliferative disorders. In a pilot study of juvenile myelomonocytic leukemia cases, we noted that CD14++/CD16- monocyte fraction was >95% in de novo juvenile myelomonocytic leukemia (JMML) with somatic PTPN11 mutations but normal in those with monosomy 7 or Noonan syndrome. Monocyte subgroup profiling by itself is not diagnostic of JMML but may distinguish molecular subgroups within JMML.

TLR7 activation in epilepsy of tuberous sclerosis complex.

BACKGROUND: Neuroinflammation and toll-like receptors (TLR) of the innate immune system have been implicated in epilepsy. We previously reported high levels of microRNAs miR-142-3p and miR-223-3p in epileptogenic brain tissue resected for the treatment of intractable epilepsy in children with tuberous sclerosis complex (TSC). As miR-142-3p has recently been reported to be a ligand and activator of TLR7, a detector of exogenous and endogenous single-stranded RNA, we evaluated TLR7 expression and downstream IL23A activation in surgically resected TSC brain tissue. METHODS: Gene expression analysis was performed on cortical tissue obtained from surgery of TSC children with pharmacoresistent epilepsy. Expression of TLRs 2, 4 and 7 was measured using NanoString nCounter assays. Real-time quantitative PCR was used to confirm TLR7 expression and compare TLR7 activation, indicated by IL-23A levels, to levels of miR-142-3p. Protein markers characteristic for TLR7 activation were assessed using data from our existing quantitative proteomics dataset of TSC tissue. Capillary electrophoresis Western blots were used to confirm TLR7 protein expression in a subset of samples. RESULTS: TLR7 transcript expression was present in all TSC specimens. The signaling competent form of TLR7 protein was detected in the membrane fraction of each sample tested. Downstream activation of TLR7 was found in epileptogenic lesions having elevated neuroinflammation indicated by clinical neuroimaging. TLR7 activity was significantly associated with tissue levels of miR-142-3p. CONCLUSION: TLR7 activation by microRNAs may contribute to the neuroinflammatory cascade in epilepsy in TSC. Further characterization of this mechanism may enable the combined of use of neuroimaging and TLR7 inhibitors in a personalized approach towards the treatment of intractable epilepsy.

Identification of a novel variant in phosphoglycerate kinase-1 (PGK1) in an African-American child (PGK1 Detroit).

The human phosphoglycerate kinase-1 enzyme is the first of two energy generating steps in the glycolysis. Since its discovery in 1968, many pathologically mutated forms of PGK1 have been described. PGK1 is expressed in all tissues. The clinical manifestations of PGK1 deficiency are some combination of anemia, central nervous system and/or musculoskeletal manifestations. We describe a case of PGK1 in an African-American child, which to our knowledge, has never been described to date. The manifestations of PGK1-Detroit (c.1105A > C (p.Thr369Pro)) include hematologic and central nervous manifestations.

A distinct microRNA expression profile is associated with α[11C]-methyl-L-tryptophan (AMT) PET uptake in epileptogenic cortical tubers resected from patients with tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is characterized by hamartomatous lesions in various organs and arises due to mutations in the TSC1 or TSC2 genes. TSC mutations lead to a range of neurological manifestations including epilepsy, cognitive impairment, autism spectrum disorders (ASD), and brain lesions that include cortical tubers. There is evidence that seizures arise at or near cortical tubers, but it is unknown why some tubers are epileptogenic while others are not. We have previously reported increased tryptophan metabolism measured with α[11C]-methyl-l-tryptophan (AMT) positron emission tomography (PET) in epileptogenic tubers in approximately two-thirds of patients with tuberous sclerosis and intractable epilepsy. However, the underlying mechanisms leading to seizure onset in TSC remain poorly characterized. MicroRNAs are enriched in the brain and play important roles in neurodevelopment and brain function. Recent reports have shown aberrant microRNA expression in epilepsy and TSC. In this study, we performed microRNA expression profiling in brain specimens obtained from TSC patients undergoing epilepsy surgery for intractable epilepsy. Typically, in these resections several non-seizure onset tubers are resected together with the seizure-onset tubers because of their proximity. We directly compared seizure onset tubers, with and without increased tryptophan metabolism measured with PET, and non-onset tubers to assess the role of microRNAs in epileptogenesis associated with these lesions. Whether a particular tuber was epileptogenic or non-epileptogenic was determined with intracranial electrocorticography, and tryptophan metabolism was measured with AMT PET. We identified a set of five microRNAs (miR-142-3p, 142-5p, 223-3p, 200b-3p and 32-5p) that collectively distinguish among the three primary groups of tubers: non-onset/AMT-cold (NC), onset/AMT-cold (OC), and onset/AMT-hot (OH). These microRNAs were significantly upregulated in OH tubers compared to the other two groups, and microRNA expression was most significantly associated with AMT-PET uptake. The microRNAs target a group of genes enriched for synaptic signaling and epilepsy risk, including SLC12A5, SYT1, GRIN2A, GRIN2B, KCNB1, SCN2A, TSC1, and MEF2C. We confirmed the interaction between miR-32-5p and SLC12A5 using a luciferase reporter assay. Our findings provide a new avenue for subsequent mechanistic studies of tuber epileptogenesis in TSC.

Altered metabolomic-genomic signature: A potential noninvasive biomarker of epilepsy.

OBJECTIVE: This study aimed to identify noninvasive biomarkers of human epilepsy that can reliably detect and localize epileptic brain regions. Having noninvasive biomarkers would greatly enhance patient diagnosis, patient monitoring, and novel therapy development. At the present time, only surgically invasive, direct brain recordings are capable of detecting these regions with precision, which severely limits the pace and scope of both clinical management and research progress in epilepsy. METHODS: We compared high versus low or nonspiking regions in nine medically intractable epilepsy surgery patients by performing integrated metabolomic-genomic-histological analyses of electrically mapped human cortical regions using high-resolution magic angle spinning proton magnetic resonance spectroscopy, cDNA microarrays, and histological analysis. RESULTS: We found a highly consistent and predictive metabolite logistic regression model with reduced lactate and increased creatine plus phosphocreatine and choline, suggestive of a chronically altered metabolic state in epileptic brain regions. Linking gene expression, cellular, and histological differences to these key metabolites using a hierarchical clustering approach predicted altered metabolic vascular coupling in the affected regions. Consistently, these predictions were validated histologically, showing both neovascularization and newly discovered, millimeter-sized microlesions. SIGNIFICANCE: Using a systems biology approach on electrically mapped human cortex provides new evidence for spatially segregated, metabolic derangements in both neurovascular and synaptic architecture in human epileptic brain regions that could be a noninvasively detectable biomarker of epilepsy. These findings both highlight the immense power of a systems biology approach and identify a potentially important role that magnetic resonance spectroscopy can play in the research and clinical management of epilepsy.

Predicting novel histopathological microlesions in human epileptic brain through transcriptional clustering.

Although epilepsy is associated with a variety of abnormalities, exactly why some brain regions produce seizures and others do not is not known. We developed a method to identify cellular changes in human epileptic neocortex using transcriptional clustering. A paired analysis of high and low spiking tissues recorded in vivo from 15 patients predicted 11 cell-specific changes together with their 'cellular interactome'. These predictions were validated histologically revealing millimetre-sized 'microlesions' together with a global increase in vascularity and microglia. Microlesions were easily identified in deeper cortical layers using the neuronal marker NeuN, showed a marked reduction in neuronal processes, and were associated with nearby activation of MAPK/CREB signalling, a marker of epileptic activity, in superficial layers. Microlesions constitute a common, undiscovered layer-specific abnormality of neuronal connectivity in human neocortex that may be responsible for many 'non-lesional' forms of epilepsy. The transcriptional clustering approach used here could be applied more broadly to predict cellular differences in other brain and complex tissue disorders.

Exosomes in Epilepsy of Tuberous Sclerosis Complex: Carriers of Pro-Inflammatory MicroRNAs.

Exosomes are a class of small, secreted extracellular vesicles (EV) that have recently gained considerable attention for their role in normal cellular function, disease processes and potential as biomarkers. Exosomes serve as intercellular messengers and carry molecular cargo that can alter gene expression and the phenotype of recipient cells. Here, we investigated alterations of microRNA cargo in exosomes secreted by epileptogenic tissue in tuberous sclerosis complex (TSC), a multi-system genetic disorder that includes brain lesions known as tubers. Approximately 90% of TSC patients suffer from seizures that originate from tubers, and ~60% are resistant to antiseizure drugs. It is unknown why some tubers cause seizures while others do not, and the molecular basis of drug-resistant epilepsy is not well understood. It is believed that neuroinflammation is involved, and characterization of this mechanism may be key to disrupting the "vicious cycle" between seizures, neuroinflammation, and increased seizure susceptibility. We isolated exosomes from epileptogenic and non-epileptogenic TSC tubers, and we identified differences in their microRNA cargo using small RNA-seq. We identified 12 microRNAs (including miR-142-3p, miR-223-3p and miR-21-5p) that are significantly increased in epileptogenic tubers and contain nucleic acid motifs that activate toll-like receptors (TLR7/8), initiating a neuroinflammatory cascade. Exosomes from epileptogenic tissue caused induction of key pathways in cultured cells, including innate immune signaling (TLR), inflammatory response and key signaling nodes SQSTM1 (p62) and CDKN1A (p21). Genes induced in vitro were also significantly upregulated in epileptogenic tissue. These results provide new evidence on the role of exosomes and non-coding RNA cargo in the neuroinflammatory cascade of epilepsy and may help advance the development of novel biomarkers and therapeutic approaches for the treatment of drug-resistant epilepsy.

Clinical Diagnosis of Red Cell Membrane Disorders: Comparison of Osmotic Gradient Ektacytometry and Eosin Maleimide (EMA) Fluorescence Test for Red Cell Band 3 (AE1, SLC4A1) Content for Clinical Diagnosis.

The measurement of band 3 (AE1, SLC4A1, CD233) content of red cells by eosin-5- maleimide (EMA) staining is swiftly replacing conventional osmotic fragility (OF) test as a tool for laboratory confirmation of hereditary spherocytosis across the globe. Our group has systematically evaluated the EMA test as a method to screen for a variety of anemias in the last 10 years, and compared these results to those obtained with the osmotic gradient ektacytometry (osmoscans) which we have used over three decades. Our overall experience allowed us to characterize the distinctive patterns with the two tests in several congenital erythrocyte membrane disorders, such as hereditary spherocytosis (HS), hereditary elliptocytosis (HE), Southeast Asian Ovalocytosis (SAO), hereditary pyropoikilocytosis (HPP) variants, erythrocyte volume disorders, various red cell enzymopathies, and hemoglobinopathies. A crucial difference between the two methodologies is that osmoscans measure red blood cell deformability of the entire sample of RBCs, while the EMA test examines the band 3 content of individual RBCs. EMA content is influenced by cell size as smaller red cells have lower amount of total membrane than larger cells. The SAO mutation alters the EMA binding site resulting in a lower EMA MCF even as the band 3 content itself is unchanged. Thus, EMA scan results should be interpreted with caution and both the histograms and dot plots should be analyzed in the context of the clinical picture and morphology.

Interictal spike connectivity in human epileptic neocortex.

OBJECTIVE: Interictal spikes are a biomarker of epilepsy, yet their precise roles are poorly understood. Using long-term neocortical recordings from epileptic patients, we investigated the spatial-temporal propagation patterns of interictal spiking. METHODS: Interictal spikes were detected in 10 epileptic patients. Short time direct directed transfer function was used to map the spatial-temporal patterns of interictal spike onset and propagation across different cortical topographies. RESULTS: Each patient had unique interictal spike propagation pattern that was highly consistent across times, regardless of the frequency band. High spiking brain regions were often not spike onset regions. We observed frequent spike propagations to shorter distances and that the central sulcus forms a strong barrier to spike propagation. Spike onset and seizure onset seemed to be distinct networks in most cases. CONCLUSIONS: Patients in epilepsy have distinct and unique network of causal propagation pattern which are very consistent revealing the underlying epileptic network. Although spike are epileptic biomarkers, spike origin and seizure onset seems to be distinct in most cases. SIGNIFICANCE: Understanding patterns of interictal spike propagation could lead to the identification patient-specific epileptic networks amenable to surgical or other treatments.

Neuroinflammatory Nexus of Pediatric Epilepsy.

A rapidly growing body of evidence supports the premise that neuroinflammation plays an important role in initiating and sustaining seizures in a range of pediatric epilepsies. Clinical and experimental evidence indicate that neuroinflammation is both an outcome and a contributor to seizures. In this manner, seizures that arise from an initial insult (e.g. infection, trauma, genetic mutation) contribute to an inflammatory response that subsequently promotes recurrent seizures. This cyclical relationship between seizures and neuroinflammation has been described as a 'vicious cycle.' Studies of human tissue resected for surgical treatment of refractory epilepsy have reported activated inflammatory and immune signaling pathways, while animal models have been used to demonstrate that key inflammatory mediators lead to increased seizure susceptibility. Further characterization of the molecular mechanisms involved in this cycle may ultimately enable the development of new therapeutic approaches for the treatment of epilepsy. In this brief review we focus on key inflammatory mediators that have become prominent in recent literature of epilepsy, including newly characterized microRNAs and their potential role in neuroinflammatory signaling.

Proteomic analysis of human epileptic neocortex predicts vascular and glial changes in epileptic regions.

Epilepsy is a common neurological disorder, which is not well understood at the molecular level. Exactly why some brain regions produce epileptic discharges and others do not is not known. Patients who fail to respond to antiseizure medication (refractory epilepsy) can benefit from surgical removal of brain regions to reduce seizure frequency. The tissue removed in these surgeries offers an invaluable resource to uncover the molecular and cellular basis of human epilepsy. Here, we report a proteomic study to determine whether there are common proteomic patterns in human brain regions that produce epileptic discharges. We analyzed human brain samples, as part of the Systems Biology of Epilepsy Project (SBEP). These brain pieces are in vivo electrophysiologically characterized human brain samples withdrawn from the neocortex of six patients with refractory epilepsy. This study is unique in that for each of these six patients the comparison of protein expression was made within the same patient: a more epileptic region was compared to a less epileptic brain region. The amount of epileptic activity was defined for each patient as the frequency of their interictal spikes (electric activity between seizures that is a parameter strongly linked to epilepsy). Proteins were resolved from three subcellular fractions, using a 2D differential gel electrophoresis (2D-DIGE), revealing 31 identified protein spots that changed significantly. Interestingly, glial fibrillary acidic protein (GFAP) was found to be consistently down regulated in high spiking brain tissue and showed a strong negative correlation with spike frequency. We also developed a two-step analysis method to select for protein species that changed frequently among the patients and identified these proteins. A total of 397 protein spots of interest (SOI) were clustered by protein expression patterns across all samples. These clusters were used as markers and this analysis predicted proteomic changes due to both histological differences and molecular pathways, revealed by examination of gene ontology clusters. Our experimental design and proteomic data analysis predicts novel glial changes, increased angiogenesis, and changes in cytoskeleton and neuronal projections between high and low interictal spiking regions. Quantitative histological staining of these same tissues for both the vascular and glial changes confirmed these findings, which provide new insights into the structural and functional basis of neocortical epilepsy.

Activity-dependent human brain coding/noncoding gene regulatory networks.

While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The brain-derived neurotrophic factor (BDNF) gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long noncoding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and noncoding human transcription using a custom lncRNA microarray identified 1288 differentially expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional 8 were adjacent to or overlapping with differentially expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, composing an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF-BDNFOS co-expression that was not achieved with a single depolarization. RNAi-mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-messenger RNA pairs validated the effect of chronic neuronal activity on the transcriptome and implied various lncRNA regulatory mechanisms. lncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity.

Elongating RNA polymerase II is disassembled through specific degradation of its largest but not other subunits in response to DNA damage in vivo.

Although previous biochemical studies have demonstrated global degradation of the largest subunit, Rpb1p, of RNA polymerase II in response to DNA damage, it is still not clear whether the initiating or elongating form of Rpb1p is targeted for degradation in vivo. Further, whether other components of RNA polymerase II are degraded in response to DNA damage remains unknown. Here, we show that the Rpb1p subunit of the elongating, but not initiating, form of RNA polymerase II is degraded at the active genes in response to 4-nitroquinoline-1-oxide-induced DNA damage in Saccharomyces cerevisiae. However, other subunits of RNA polymerase II are not degraded in response to DNA damage. Further, we show that Rpb1p is essential for RNA polymerase II assembly at the active gene, and thus, the degradation of Rpb1p following DNA damage disassembles elongating RNA polymerase II. Taken together, our data demonstrate that Rpb1p but not other subunits of elongating RNA polymerase II is specifically degraded in response to DNA damage, and such a degradation of Rpb1p is critical for the disassembly of elongating RNA polymerase II at the DNA lesion in vivo.