Elizabeth A. Thiele

Mutational Analysis in a Cohort of 224 Tuberous Sclerosis Patients Indicates Increased Severity of TSC2, Compared with TSC1, Disease in Multiple Organs

Tuberous sclerosis (TSC) is a relatively common hamartoma syndrome caused by mutations in either of two genes, TSC1 and TSC2. Here we report comprehensive mutation analysis in 224 index patients with TSC and correlate mutation findings with clinical features. Denaturing high-performance liquid chromatography, long-range polymerase chain reaction (PCR), and quantitative PCR were used for mutation detection. Mutations were identified in 186 (83%) of 224 of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had, on average, milder disease in comparison with patients with TSC2 mutations, despite being of similar age. They had a lower frequency of seizures and moderate-to-severe mental retardation, fewer subependymal nodules and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiofibroma. Patients in whom no mutation was found also had disease that was milder, on average, than that in patients with TSC2 mutations and was somewhat distinct from patients with TSC1 mutations. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forehead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear to be less common in TSC1 than in TSC2. The reduced severity of disease in patients without defined mutations suggests that many of these patients are mosaic for a TSC2 mutation and/or have TSC because of mutations in an as-yet-unidentified locus with a relatively mild clinical phenotype.

Nerve Growth Factor Activates Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways To Stimulate CREB Serine 133 Phosphorylation

ABSTRACT The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

Efficacy and safety of everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis complex (EXIST-1): a multicentre, randomised, placebo-controlled phase 3 trial

BACKGROUND Tuberous sclerosis complex is a genetic disorder leading to constitutive activation of mammalian target of rapamycin (mTOR) and growth of benign tumours in several organs. In the brain, growth of subependymal giant cell astrocytomas can cause life-threatening symptoms--eg, hydrocephalus, requiring surgery. In an open-label, phase 1/2 study, the mTOR inhibitor everolimus substantially and significantly reduced the volume of subependymal giant cell astrocytomas. We assessed the efficacy and safety of everolimus in patients with subependymal giant cell astrocytomas associated with tuberous sclerosis complex. METHODS In this double-blind, placebo-controlled, phase 3 trial, patients (aged 0-65 years) in 24 centres in Australia, Belgium, Canada, Germany, the UK, Italy, the Netherlands, Poland, Russian Federation, and the USA were randomly assigned, with an interactive internet-response system, in a 2:1 ratio to oral everolimus 4·5 mg/m(2) per day (titrated to achieve blood trough concentrations of 5-15 ng/mL) or placebo. Eligible patients had a definite diagnosis of tuberous sclerosis complex and at least one lesion with a diameter of 1 cm or greater, and either serial growth of a subependymal giant cell astrocytoma, a new lesion of 1 cm or greater, or new or worsening hydrocephalus. The primary endpoint was the proportion of patients with confirmed response--ie, reduction in target volume of 50% or greater relative to baseline in subependymal giant cell astrocytomas. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00789828. FINDINGS 117 patients were randomly assigned to everolimus (n=78) or placebo (n=39). 27 (35%) patients in the everolimus group had at least 50% reduction in the volume of subependymal giant cell astrocytomas versus none in the placebo group (difference 35%, 95% CI 15-52; one-sided exact Cochran-Mantel-Haenszel test, p<0·0001). Adverse events were mostly grade 1 or 2; no patients discontinued treatment because of adverse events. The most common adverse events were mouth ulceration (25 [32%] in the everolimus group vs two [5%] in the placebo group), stomatitis (24 [31%] vs eight [21%]), convulsion (18 [23%] vs ten [26%]), and pyrexia (17 [22%] vs six [15%]). INTERPRETATION These results support the use of everolimus for subependymal giant cell astrocytomas associated with tuberous sclerosis. Additionally, everolimus might represent a disease-modifying treatment for other aspects of tuberous sclerosis. FUNDING Novartis Pharmaceuticals.

Cannabidiol: Pharmacology and potential therapeutic role in epilepsy and other neuropsychiatric disorders

To present a summary of current scientific evidence about the cannabinoid, cannabidiol (CBD) with regard to its relevance to epilepsy and other selected neuropsychiatric disorders. We summarize the presentations from a conference in which invited participants reviewed relevant aspects of the physiology, mechanisms of action, pharmacology, and data from studies with animal models and human subjects. Cannabis has been used to treat disease since ancient times. Δ9‐Tetrahydrocannabinol (Δ9‐THC) is the major psychoactive ingredient and CBD is the major nonpsychoactive ingredient in cannabis. Cannabis and Δ9‐THC are anticonvulsant in most animal models but can be proconvulsant in some healthy animals. The psychotropic effects of Δ9‐THC limit tolerability. CBD is anticonvulsant in many acute animal models, but there are limited data in chronic models. The antiepileptic mechanisms of CBD are not known, but may include effects on the equilibrative nucleoside transporter; the orphan G‐protein‐coupled receptor GPR55; the transient receptor potential of vanilloid type‐1 channel; the 5‐HT1a receptor; and the α3 and α1 glycine receptors. CBD has neuroprotective and antiinflammatory effects, and it appears to be well tolerated in humans, but small and methodologically limited studies of CBD in human epilepsy have been inconclusive. More recent anecdotal reports of high‐ratio CBD:Δ9‐THC medical marijuana have claimed efficacy, but studies were not controlled. CBD bears investigation in epilepsy and other neuropsychiatric disorders, including anxiety, schizophrenia, addiction, and neonatal hypoxic‐ischemic encephalopathy. However, we lack data from well‐powered double‐blind randomized, controlled studies on the efficacy of pure CBD for any disorder. Initial dose‐tolerability and double‐blind randomized, controlled studies focusing on target intractable epilepsy populations such as patients with Dravet and Lennox‐Gastaut syndromes are being planned. Trials in other treatment‐resistant epilepsies may also be warranted.

The natural history of epilepsy in tuberous sclerosis complex

Background:  Although epilepsy affects most patients with tuberous sclerosis complex (TSC), little is known about the natural history of epilepsy in this genetic disease.

Subependymal giant cell tumors in tuberous sclerosis complex

Objectives: To describe the clinical presentations, radiologic features, and postoperative outcomes of a clinic-based population of patients with subependymal giant cell tumors (SGCT) and tuberous sclerosis complex (TSC) and to redefine and reclassify SGCT based on radiologic, clinical, and pathologic criteria. Methods: Of 134 TSC patients evaluated from December 2001 to November 2003, 11 (8.2%) had undergone resection of a pathologically confirmed SGCT. The authors reviewed the medical records of each case. Follow-up ranged from 2 months to 36 years. Results: Four were asymptomatic at the time of resection while the other seven presented subacutely with fatigue, decreased appetite, headache, increased seizure frequency, visual field deficit, cognitive decline, or behavioral problems. Poor outcomes occurred in all patients aged 11 years or older at the time of resection. Conclusions: Subependymal giant cell tumors in patients with TSC appear to be of mixed glioneuronal lineage, and, therefore, the current practice of classifying these tumors as astrocytomas merits revision. The clinical diagnosis of SGCT should be made for subependymal lesions in TSC that are associated with symptoms, papilledema, or radiologic evidence of hydrocephalus or interval growth. A diagnosis of probable SGCT should be made when a lesion has the potential to cause obstruction based on size or location. Annual screening by MRI with or without contrast is indicated until at least 21 years of age even if subependymal nodules are absent on initial imaging. A diagnosis of SGCT or probable SGCT warrants more frequent monitoring or surgical intervention.

Low-glycemic-index treatment: A liberalized ketogenic diet for treatment of intractable epilepsy

The ketogenic diet is often effective for intractable epilepsy, but many patients have trouble complying with the strict regimen. The authors tested an alternative diet regimen, a low-glycemic-index treatment, with more liberal total carbohydrate intake but restricted to foods that produce relatively little increase in blood glucose (glycemic index < 50). Ten of 20 patients treated with this regimen experienced a greater than 90% reduction in seizure frequency.

Managing epilepsy in tuberous sclerosis complex.

Epilepsy is very common in tuberous sclerosis complex and occurs in 80 to 90% of affected individuals during their lifetime. Onset usually occurs during childhood, and up to one third of children with tuberous sclerosis complex will develop infantile spasms. Although not completely understood, the incidence of epilepsy is thought to relate to the neuropathologic features of the disorder, including cortical tubers and other dysgenetic features. Individuals with tuberous sclerosis complex frequently have epileptiform features to their electroencephalograms. Treatment of epilepsy in tuberous sclerosis complex is similar to epilepsy resulting from other causes and includes anticonvulsant medications, the vagus nerve stimulator, and the ketogenic diet. Vigabatrin has been shown to be particularly effective in treating infantile spasms in the setting of tuberous sclerosis complex. Epilepsy surgery has a very important role in the management of children and adults with pharmacoresistant epilepsy in tuberous sclerosis complex. (J Child Neurol 2004;19:680—686).

Multicenter Phase 2 Trial of Sirolimus for Tuberous Sclerosis: Kidney Angiomyolipomas and Other Tumors Regress and VEGF- D Levels Decrease

Background Tuberous sclerosis (TSC) related tumors are characterized by constitutively activated mTOR signaling due to mutations in TSC1 or TSC2. Methods We completed a phase 2 multicenter trial to evaluate the efficacy and tolerability of the mTOR inhibitor, sirolimus, for the treatment of kidney angiomyolipomas. Results 36 adults with TSC or TSC/LAM were enrolled and started on daily sirolimus. The overall response rate was 44.4% (95% confidence intervals [CI] 28 to 61); 16/36 had a partial response. The remainder had stable disease (47.2%, 17/36), or were unevaluable (8.3%, 3/36). The mean decrease in kidney tumor size (sum of the longest diameters [sum LD]) was 29.9% (95% CI, 22 to 37; n = 28 at week 52). Drug related grade 1–2 toxicities that occurred with a frequency of >20% included: stomatitis, hypertriglyceridemia, hypercholesterolemia, bone marrow suppression (anemia, mild neutropenia, leucopenia), proteinuria, and joint pain. There were three drug related grade 3 events: lymphopenia, headache, weight gain. Kidney angiomyolipomas regrew when sirolimus was discontinued but responses tended to persist if treatment was continued after week 52. We observed regression of brain tumors (SEGAs) in 7/11 cases (26% mean decrease in diameter), regression of liver angiomyolipomas in 4/5 cases (32.1% mean decrease in longest diameter), subjective improvement in facial angiofibromas in 57%, and stable lung function in women with TSC/LAM (n = 15). A correlative biomarker study showed that serum VEGF-D levels are elevated at baseline, decrease with sirolimus treatment, and correlate with kidney angiomyolipoma size (Spearman correlation coefficient 0.54, p = 0.001, at baseline). Conclusions Sirolimus treatment for 52 weeks induced regression of kidney angiomyolipomas, SEGAs, and liver angiomyolipomas. Serum VEGF-D may be a useful biomarker for monitoring kidney angiomyolipoma size. Future studies are needed to determine benefits and risks of longer duration treatment in adults and children with TSC. Trial Registration Clinicaltrials.gov NCT00126672

Trial of Cannabidiol for Drug-Resistant Seizures in the Dravet Syndrome

BACKGROUND The Dravet syndrome is a complex childhood epilepsy disorder that is associated with drug‐resistant seizures and a high mortality rate. We studied cannabidiol for the treatment of drug‐resistant seizures in the Dravet syndrome. METHODS In this double‐blind, placebo‐controlled trial, we randomly assigned 120 children and young adults with the Dravet syndrome and drug‐resistant seizures to receive either cannabidiol oral solution at a dose of 20 mg per kilogram of body weight per day or placebo, in addition to standard antiepileptic treatment. The primary end point was the change in convulsive‐seizure frequency over a 14‐week treatment period, as compared with a 4‐week baseline period. RESULTS The median frequency of convulsive seizures per month decreased from 12.4 to 5.9 with cannabidiol, as compared with a decrease from 14.9 to 14.1 with placebo (adjusted median difference between the cannabidiol group and the placebo group in change in seizure frequency, ‐22.8 percentage points; 95% confidence interval [CI], ‐41.1 to ‐5.4; P=0.01). The percentage of patients who had at least a 50% reduction in convulsive‐seizure frequency was 43% with cannabidiol and 27% with placebo (odds ratio, 2.00; 95% CI, 0.93 to 4.30; P=0.08). The patients overall condition improved by at least one category on the seven‐category Caregiver Global Impression of Change scale in 62% of the cannabidiol group as compared with 34% of the placebo group (P=0.02). The frequency of total seizures of all types was significantly reduced with cannabidiol (P=0.03), but there was no significant reduction in nonconvulsive seizures. The percentage of patients who became seizure‐free was 5% with cannabidiol and 0% with placebo (P=0.08). Adverse events that occurred more frequently in the cannabidiol group than in the placebo group included diarrhea, vomiting, fatigue, pyrexia, somnolence, and abnormal results on liver‐function tests. There were more withdrawals from the trial in the cannabidiol group. CONCLUSIONS Among patients with the Dravet syndrome, cannabidiol resulted in a greater reduction in convulsive‐seizure frequency than placebo and was associated with higher rates of adverse events. (Funded by GW Pharmaceuticals; ClinicalTrials.gov number, NCT02091375.)