Matthew R. Fleming

De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy.

Malignant migrating partial seizures of infancy (MMPSI) is a rare epileptic encephalopathy of infancy that combines pharmacoresistant seizures with developmental delay. We performed exome sequencing in three probands with MMPSI and identified de novo gain-of-function mutations affecting the C-terminal domain of the KCNT1 potassium channel. We sequenced KCNT1 in 9 additional individuals with MMPSI and identified mutations in 4 of them, in total identifying mutations in 6 out of 12 unrelated affected individuals. Functional studies showed that the mutations led to constitutive activation of the channel, mimicking the effects of phosphorylation of the C-terminal domain by protein kinase C. In addition to regulating ion flux, KCNT1 has a non-conducting function, as its C terminus interacts with cytoplasmic proteins involved in developmental signaling pathways. These results provide a focus for future diagnostic approaches and research for this devastating condition.

Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis

In severe early-onset epilepsy, precise clinical and molecular genetic diagnosis is complex, as many metabolic and electro-physiological processes have been implicated in disease causation. The clinical phenotypes share many features such as complex seizure types and developmental delay. Molecular diagnosis has historically been confined to sequential testing of candidate genes known to be associated with specific sub-phenotypes, but the diagnostic yield of this approach can be low. We conducted whole-genome sequencing (WGS) on six patients with severe early-onset epilepsy who had previously been refractory to molecular diagnosis, and their parents. Four of these patients had a clinical diagnosis of Ohtahara Syndrome (OS) and two patients had severe non-syndromic early-onset epilepsy (NSEOE). In two OS cases, we found de novo non-synonymous mutations in the genes KCNQ2 and SCN2A. In a third OS case, WGS revealed paternal isodisomy for chromosome 9, leading to identification of the causal homozygous missense variant in KCNT1, which produced a substantial increase in potassium channel current. The fourth OS patient had a recessive mutation in PIGQ that led to exon skipping and defective glycophosphatidyl inositol biosynthesis. The two patients with NSEOE had likely pathogenic de novo mutations in CBL and CSNK1G1, respectively. Mutations in these genes were not found among 500 additional individuals with epilepsy. This work reveals two novel genes for OS, KCNT1 and PIGQ. It also uncovers unexpected genetic mechanisms and emphasizes the power of WGS as a clinical tool for making molecular diagnoses, particularly for highly heterogeneous disorders.

Regulation of neuronal excitability by interaction of Fragile X Mental Retardation Protein with Slack potassium channels

Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na+-activated K+ channels (KNa), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing native Aplysia Slack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel–FMRP interactions may link changes in neuronal firing to changes in protein translation.

Use of optical biosensors to detect modulation of Slack potassium channels by G protein-coupled receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ion to flow through the plasma membrane. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex. Traditional assays examining the interaction between channels and regulatory proteins generally provide little information on the time-course of interactions in living cells. We have now used a novel label-free technology to detect changes in the distribution of mass close to the plasma membrane following modulation of potassium channels by G protein-coupled receptors (GPCRs). This technology uses optical sensors embedded in microplates to detect changes in the refractive index at the surface of cells. Although the activation of GPCRs has been studied with this system, protein-protein interactions due to modulation of ion channels have not yet been characterized. Here we present data that the characteristic pattern of mass distribution following GPCR activation is significantly modified by the presence of a sodium-activated potassium channel, Slack-B, a channel that is known to be potently modulated by activation of these receptors.

Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating

Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane(1). To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex(2,3). Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the proteins behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Stimulation of Slack K+ Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex

Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.

Recurrent Falls Among Elderly Patients and the Impact of Anticoagulation Therapy

BackgroundFalls are the leading source of injury and trauma-related hospital admissions for elderly adults in the USA. Elderly patients with a history of a fall have the highest risk of falling again, and the decision on whether to continue anticoagulation after a fall is difficult. To inform this decision, we evaluated the rate of recurrent falls and the impact of anticoagulation on outcomes.MethodsAll patients of age  ≥ 65 years and hospitalized for a fall in the first 6 months of 2013 and 2014 were identified in the nationwide readmission database, a nationally representative all-payer database tracking patient readmissions. Readmissions for a recurrent fall within 6 months, and mortality and bleeding injuries (intracranial hemorrhage, solid organ bleed, and hemothorax) during readmission were identified. Logistic regression evaluated factors associated with mortality on repeat falls.ResultsOf the 331,982 patients admitted for a fall, 15,565 (4.7%) were admitted for a recurrent fall within 6 months. The median time to repeat fall was 57 days (IQR 19–111 days), and 9.0% (1406) of repeat fallers were on anticoagulation. The rate of bleeding injury was similar regardless of anticoagulation status (12.8 vs. 12.7% not on anticoagulation, p = 0.97); however, among patients with a bleeding injury, those on anticoagulation had significantly higher mortality (21.5 vs. 6.9% not on anticoagulation, p < 0.01).ConclusionAmong patients hospitalized for a fall, 4.7% will be hospitalized for a recurrent fall within 6 months. Patients on anticoagulation with repeat falls do not have increased rates of bleeding injury but do have significantly higher rates of death with a bleeding injury. This information is essential to discuss with patients when deciding to restart their anticoagulation.

Socially Responsible Surgery: Building Recognition and Coalition

Importance Socially responsible surgery (SRS) integrates surgery and public health, providing a framework for research, advocacy, education, and clinical practice to address the social barriers of health that decrease surgical access and worsen surgical outcomes in underserved patient populations. These patients face disparities in both health and in health care, which can be effectively addressed by surgeons in collaboration with allied health professionals. Objective We reviewed the current state of surgical access and outcomes of underserved populations in American rural communities, American urban communities, and in low- and middle-income countries. Evidence review We searched PubMed using standardized search terms and reviewed the reference lists of highly relevant articles. We reviewed the reports of two recent global surgery commissions. Conclusion There is an opportunity for scholarship in rural surgery, urban surgery, and global surgery to be unified under the concept of SRS. The burden of surgical disease and the challenges to management demonstrate that achieving optimal health outcomes requires more than excellent perioperative care. Surgeons can and should regularly address the social determinants of health experienced by their patients. Formalized research and training opportunities are needed to meet the growing enthusiasm among surgeons and trainees to develop their practice as socially responsible surgeons.

Surgical referral coordination from a first-level hospital: A prospective case study from rural Nepal

BackgroundPatients in isolated rural communities typically lack access to surgical care. It is not feasible for most rural first-level hospitals to provide a full suite of surgical specialty services. Comprehensive surgical care thus depends on referral systems. There is minimal literature, however, on the functioning of such systems.MethodsWe undertook a prospective case study of the referral and care coordination process for cardiac, orthopedic, plastic, gynecologic, and general surgical conditions at a district hospital in rural Nepal from 2012 to 2014. We assessed the referral process using the World Health Organization’s Health Systems Framework.ResultsWe followed the initial 292 patients referred for surgical services in the program. 152 patients (52%) received surgery and four (1%) suffered a complication (three deaths and one patient reported complication). The three most common types of surgery performed were: orthopedics (43%), general (32%), and plastics (10%). The average direct and indirect cost per patient referred, including food, transportation, lodging, medications, diagnostic examinations, treatments, and human resources was US