Scott M. Paul

Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus

Positive-strand RNA viruses are the largest virus class and include many pathogens such as hepatitis C virus and the severe acute respiratory syndrome coronavirus (SARS). Brome mosaic virus (BMV) is a representative positive-strand RNA virus whose RNA replication, gene expression, and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. By using traditional yeast genetics, host genes have been identified that function in controlling BMV translation, selecting BMV RNAs as replication templates, activating the replication complex, maintaining a lipid composition required for membrane-associated RNA replication, and other steps. To more globally and systematically identify such host factors, we used engineered BMV derivatives to assay viral RNA replication in each strain of an ordered, genome-wide set of yeast single-gene deletion mutants. Each deletion strain was transformed to express BMV replicase proteins and a BMV RNA replication template with the capsid gene replaced by a luciferase reporter. Luciferase expression, which is dependent on viral RNA replication and RNA-dependent mRNA synthesis, was measured in intact yeast cells. Approximately 4,500 yeast deletion strains (≈80% of yeast genes) were screened in duplicate and selected strains analyzed further. This functional genomics approach revealed nearly 100 genes whose absence inhibited or stimulated BMV RNA replication and/or gene expression by 3- to >25-fold. Several of these genes were shown previously to function in BMV replication, validating the approach. Newly identified genes include some in RNA, protein, or membrane modification pathways and genes of unknown function. The results further illuminate virus and cell pathways. Further refinement of virus screening likely will reveal contributions from additional host genes.

Sustained Response and Prevention of Damage Progression in Patients With Neonatal-Onset Multisystem Inflammatory Disease Treated With Anakinra: A Cohort Study to Determine Three- and Five-Year Outcomes

OBJECTIVE Blocking interleukin-1 with anakinra in patients with the autoinflammatory syndrome neonatal-onset multisystem inflammatory disease (NOMID) reduces systemic and organ-specific inflammation. However, the impact of long-term treatment has not been established. This study was undertaken to evaluate the long-term effect of anakinra on clinical and laboratory outcomes and safety in patients with NOMID. METHODS We conducted a cohort study of 26 NOMID patients ages 0.80-42.17 years who were followed up at the NIH and treated with anakinra 1-5 mg/kg/day for at least 36 months. Disease activity was assessed using daily diaries, questionnaires, and C-reactive protein level. Central nervous system (CNS) inflammation, hearing, vision, and safety were evaluated. RESULTS Sustained improvements in diary scores, parents/patients and physicians global scores of disease activity, parents/patients pain scores, and inflammatory markers were observed (all P<0.001 at 36 and 60 months). At 36 and 60 months, CNS inflammation was suppressed, with decreased cerebrospinal fluid white blood cell counts (P=0.0026 and P=0.0076, respectively), albumin levels, and opening pressures (P=0.0012 and P<0.001, respectively). Most patients showed stable or improved hearing. Cochlear enhancement on magnetic resonance imaging correlated with continued hearing loss. Visual acuity and peripheral vision were stable. Low optic nerve size correlated with poor visual field. Bony lesions progressed. Adverse events other than viral infections were rare, and all patients continued to receive the medication. CONCLUSION These findings indicate that anakinra provides sustained efficacy in the treatment of NOMID for up to 5 years, with the requirement of dose escalation. Damage progression in the CNS, ear, and eye, but not bone, is preventable. Anakinra is well tolerated overall.

Severe congenital neutropenia resulting from G6PC3 deficiency with increased neutrophil CXCR4 expression and myelokathexis

Mutations in more than 15 genes are now known to cause severe congenital neutropenia (SCN); however, the pathologic mechanisms of most genetic defects are not fully defined. Deficiency of G6PC3, a glucose-6-phosphatase, causes a rare multisystem syndrome with SCN first described in 2009. We identified a family with 2 children with homozygous G6PC3 G260R mutations, a loss of enzymatic function, and typical syndrome features with the exception that their bone marrow biopsy pathology revealed abundant neutrophils consistent with myelokathexis. This pathologic finding is a hallmark of another type of SCN, WHIM syndrome, which is caused by gain-of-function mutations in CXCR4, a chemokine receptor and known neutrophil bone marrow retention factor. We found markedly increased CXCR4 expression on neutrophils from both our G6PC3-deficient patients and G6pc3(-/-) mice. In both patients, granulocyte colony-stimulating factor treatment normalized CXCR4 expression and neutrophil counts. In G6pc3(-/-) mice, the specific CXCR4 antagonist AMD3100 rapidly reversed neutropenia. Thus, myelokathexis associated with abnormally high neutrophil CXCR4 expression may contribute to neutropenia in G6PC3 deficiency and responds well to granulocyte colony-stimulating factor.

An approach to the identification of anomalies and etiologies in neonates with identified or suspected VACTERL (vertebral defects, anal atresia, tracheo-esophageal fistula with esophageal atresia, cardiac anomalies, renal anomalies, and limb anomalies) association

VATER association was first described in the early 1970s as the non-random co-occurrence of congenital malformations including: Vertebral defects, Anal atresia, Tracheo-Esophageal fistula (TEF) with or without esophageal atresia (EA), and Radial and Renal dysplasia.1 Following initial reports, it was suggested that “V” should include Vascular anomalies (including single umbilical artery). Cardiac malformations (“C”) and Limb (“L”) anomalies other than radial anomalies were also added, such that the term “VACTERL” became the most common descriptor, despite variable evidence for the inclusion of features such as cardiac or renal anomalies.2–6 The presence of VACTERL association, which usually requires at least 3 component features and the absence of evidence for an overlapping condition, is estimated to occur in approximately 1/10,000–1/40,000 live births.7, 8 Just as there are challenges in defining the condition, there is no standard approach for the initial diagnostic work-up of a neonate with identified or suspected VACTERL association. This can be problematic: missed manifestations may obfuscate the etiological work-up8; delay medical interventions, potentially contributing to higher morbidity and mortality9; result in less informed and effective counseling. To attempt to address these issues, we assembled a multi-disciplinary group of clinicians and researchers whose expertise focuses on VACTERL association and/or its individual component features. Following review of the literature, and based upon our collective experience, we offer suggestions for the evaluation of individuals identified or suspected to have VACTERL association. Literature Search We conducted a PubMed-based literature search for case reports and collections of patients identified or suspected of having VACTERL association and/or associated component features. Search terms included the following: Anal atresia; Anorectal malformations; Cardiac anomalies; Cardiac malformations; Cardiovascular anomalies; Cardiovascular malformations; Esophageal atresia; Genitourinary anomalies; Genitourinary malformations; Imperforate anus; Limb anomalies; Limb malformations; Radial anomalies; Radial dysplasia; Renal anomalies; Renal malformations; TEF; Tracheo-esophageal fistula; VACTERL; VATER; Vertebral anomalies; Vertebral malformations. Only articles describing human patients were considered, and articles were excluded if they did not pertain to component features specifically seen in VACTERL association.

Evaluating interventions to improve gait in cerebral palsy: a meta‐analysis of spatiotemporal measures

A number of interventions to improve gait in individuals with cerebral palsy (CP) have been reported in the literature. The aim of this study was to perform a meta‐analysis of these studies to determine the overall efficacy of these interventions. Effect sizes (Hedges g) for spatiotemporal measures of gait (velocity, cadence, stride length) pre‐ and postintervention were analyzed. Sixty‐three studies were included, and the overall effect size was statistically significant for both fixed effects and random models. Types of interventions were grouped into spasticity treatments, orthopedic (bony and soft tissue) surgery, lower extremity orthoses, or ‘other’. When the data were analyzed in subgroups by type of intervention, each intervention had a statistically significant effect size with the exception of the ‘other’. More importantly, the present study indicates the need to address participant inclusion criteria and power analysis more adequately in future research studies of interventions to improve gait in CP.

Exercise Capacity and Idebenone Intervention in Children and Adolescents With Friedreich Ataxia

OBJECTIVE To determine the exercise capacity of children and adolescents with Friedreichs Ataxia (FA) and to evaluate the effects of 6 months of idebenone treatment on exercise capacity. DESIGN Exploratory endpoint in a randomized double-blind, placebo-controlled, phase II clinical trial designed to investigate the effects of idebenone on a biomarker of oxidative stress. SETTING Exercise physiology laboratory in a single clinical research center. PARTICIPANTS Ambulatory subjects (N=48; age range, 9-17 y) with genetically confirmed FA. INTERVENTION Idebenone administered orally 3 times a day for a total daily dose of approximately 5, 15, and 45 mg/kg or matching placebo for 6 months. MAIN OUTCOME MEASURES Peak oxygen consumption per unit time (peak VO(2)) and peak work rate (WR) were measured during incremental exercise testing at baseline and after treatment. Echocardiography and neurologic assessments were also completed before and after treatment. RESULTS Baseline mean peak VO(2) +/- SD was 746+/-246 mL/min (16.2+/-5.8 mL/kg/min), and WR was 40+/-23 W for all subjects. Peak VO(2) and WR were correlated with short guanine-adenine-adenine allele length and neurologic function. Relative left ventricular wall thickness was increased but left ventricular ejection fraction was normal in most subjects; there was no relationship between any exercise and echocardiographic measures. There were no significant changes in mean peak VO(2) or WR after idebenone treatment at any dose level relative to placebo. CONCLUSIONS Exercise capacity in children and adolescents with FA was significantly impaired. The basis for the impairment appears to be multifactorial and correlated to the degree of neurologic impairment. Although idebenone has previously been shown potentially to improve features of FA, idebenone treatment did not increase exercise capacity relative to placebo.

Rehabilitation Management of Friedreich Ataxia : Lower Extremity Force-Control Variability and Gait Performance

We describe the rehabilitation management during a 12-month period of a 14-year-old female with Friedreich ataxia. Interventions included task-oriented bimanual reaching activities, functional strengthening, and gait training using a walker featuring tension-controlled wheels and a reverse-braking system. Her physical status was assessed with the Nine-Hole Peg Test, single limb stance time, manual muscle testing, self-reported falls, isometric force control testing, and 3-dimensional gait analysis in a motion-capture laboratory. Although measures of the patient’s Nine-Hole Peg Test, single limb stance time, and manual muscle testing reflected minimal changes, her gait speed decreased by 69.4%. However, the force-control targeting of her dominant knee extensors showed a 43.7% increase in force variability that was concomitant with her decline in gait performance. The decrement of her initial gait speed was reduced to 42.9% on replacing the wheeled walker with the U-Step Walking Stabilizer at the end of the intervention period. Although the patient’s gait remained significantly impaired, extended use of the U-Step Walking Stabilizer modestly improved her gait performance, and her rate of falls decreased from 10 to 3 per month. Our observations suggest that use of force-control testing as proxy measures of ataxia and tension-controlled gait aids show promise in the management of Friedreich ataxia and merit further investigation.

Bisphosphonate Treatment for Children With Disabling Conditions

Fractures are a frequent source of morbidity in children with disabling conditions. The assessment of bone density in this population is challenging, because densitometry is influenced by dynamic forces affecting the growing skeleton and may be further confounded by positioning difficulties and surgical hardware. First‐line treatment for pediatric osteoporosis involves conservative measures, including optimizing the management of underlying conditions, maintaining appropriate calcium and vitamin D intake, encouraging weight‐bearing physical activity, and monitoring measurements of bone mineral density. Bisphosphonates are a class of medications that increase bone mineral density by inhibiting bone resorption. Although bisphosphonates are commonly prescribed for treatment of adult osteoporosis, their use in pediatric patients is controversial because of the lack of long‐term safety and efficacy data.

A Mutation in the Essential Gene gmk (Encoding Guanlyate Kinase) Generates a Requirement for Adenine at Low Temperature in Salmonella enterica

In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides. Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature. Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher Km for ATP than the wild-type enzyme. The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference. We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes.

Reliability, validity, and precision of an active stereophotogrammetry system for three-dimensional evaluation of the human torso

To determine the reliability, stability, validity and precision of a stereophotogrammetry (SP) system for use in quantifying the complex three-dimensional structure of the human torso, we performed assessments of the system using images of geometric solids and a human-form mannequin. Analysis of geometric solids revealed excellent intra- and interrater reliability of the system for linear, surface area and volume measurements (r>0.99, P<0.001). Overall, no significant difference was found between SP and manual measurements (F=4.23, P>0.06). The system exhibited excellent stability in images of the mannequin over time (r>0.99). The limit of precision (error>5%) of the system to detect objects on the surface of the mannequin was estimated at an object size of 23.5cm(2) for surface area and 32mL for volume. These results demonstrate the capability of SP of the torso to be used as a reliable, stable and valid measure of torso morphology to be applied as a clinical outcome tool in studies of bony and soft tissue pathologies such as scoliosis, rib deformities, obesity or edema.