Genotype-phenotype correlation in CLCN4-related developmental and epileptic encephalopathy.

CLCN4-related disorder is a rare X-linked neurodevelopmental condition with a pathogenic mechanism yet to be elucidated. CLCN4 encodes the vesicular 2Cl-/H+ exchanger ClC-4, and CLCN4 pathogenic variants frequently result in altered ClC-4 transport activity. The precise cellular and molecular function of ClC-4 remains unknown; however, together with ClC-3, ClC-4 is thought to have a role in the ion homeostasis of endosomes and intracellular trafficking. We reviewed our research database for patients with CLCN4 variants and epilepsy, and performed thorough phenotyping. We examined the functional properties of the variants in mammalian cells using patch-clamp electrophysiology, protein biochemistry, and confocal fluorescence microscopy. Three male patients with developmental and epileptic encephalopathy were identified, with differing phenotypes. Patients #1 and #2 had normal growth parameters and normal-appearing brains on MRI, while patient #3 had microcephaly, microsomia, complete agenesis of the corpus callosum and cerebellar and brainstem hypoplasia. The p.(Gly342Arg) variant of patient #1 significantly impaired ClC-4's heterodimerization capability with ClC-3 and suppressed anion currents. The p.(Ile549Leu) variant of patient #2 and p.(Asp89Asn) variant of patient #3 both shift the voltage dependency of transport activation by 20 mV to more hyperpolarizing potentials, relative to the wild-type, with p.(Asp89Asn) favouring higher transport activity. We concluded that p.(Gly342Arg) carried by patient #1 and the p.(Ile549Leu) expressed by patient #2 impair ClC-4 transport function, while the p.(Asp89Asn) variant results in a gain-of-transport function; all three variants result in epilepsy and global developmental impairment, but with differences in epilepsy presentation, growth parameters, and presence or absence of brain malformations.

Utility of genetic testing in the pre-surgical evaluation of children with drug-resistant epilepsy.

We evaluated the utility of genetic testing in the pre-surgical evaluation of pediatric patients with drug-resistant focal epilepsy. This single-center retrospective study reviewed the charts of all pediatric patients referred for epilepsy surgery evaluation over a 5-year period. We extracted and analyzed results of genetic testing as well as clinical, EEG, and neuroimaging data. Of 125 patients referred for epilepsy surgical evaluation, 86 (69%) had some form of genetic testing. Of these, 18 (21%) had a pathogenic or likely pathogenic variant identified. Genes affected included NPRL3 (3 patients, all related), TSC2 (3 patients), KCNH1, CHRNA4, SPTAN1, DEPDC5, SCN2A, ARX, SCN1A, DLG4, and ST5. One patient had ring chromosome 20, one a 7.17p12 duplication, and one a 15q13 deletion. In six patients, suspected epileptogenic lesions were identified on brain MRI that were thought to be unrelated to the genetic finding. A specific medical therapy choice was allowed due to genetic diagnosis in three patients who did not undergo surgery. Obtaining a molecular diagnosis may dramatically alter management in pediatric patients with drug-resistant focal epilepsy. Genetic testing should be incorporated as part of standard investigations in the pre-surgical work-up of pediatric patients with drug-resistant focal epilepsy.

The phenotypic spectrum of epilepsy associated with periventricular nodular heterotopia.

BACKGROUND: Periventricular nodular heterotopia (PVNH) is a congenital brain malformation often associated with seizures. We aimed to clarify the spectrum of epilepsy phenotypes in PVNH and the significance of specific brain malformation patterns. METHODS: In this retrospective cohort study, we recruited people with PVNH and a history of seizures, and collected data via medical record review and a standardized questionnaire. RESULTS: One hundred individuals were included, aged 1 month to 61 years. Mean seizure onset age was 7.9 years. Ten patients had a self-limited epilepsy course and 35 more were pharmacoresponsive. Fifty-five had ongoing seizures, of whom 23 met criteria for drug resistance. Patients were subdivided as follows: isolated PVNH ("PVNH-Only") single nodule (18) or multiple nodules (21) and PVNH with additional brain malformations ("PVNH-Plus") single nodule (8) or multiple nodules (53). Of PVNH-Only single nodule, none had drug-resistant seizures. Amongst PVNH-Plus, 55% with multiple unilateral nodules were pharmacoresponsive, compared to only 21% with bilateral nodules. PVNH-Plus with bilateral nodules demonstrated the highest proportion of drug resistance (39%). A review of genetic testing results revealed eight patients with pathogenic or likely pathogenic single-gene variants, two of which were FLNA. Five had copy number variants, two of which were pathogenic. CONCLUSIONS: The spectrum of epilepsy phenotypes in PVNH is broad, and seizure patterns are variable; however, epilepsy course may be predicted to an extent by the pattern of malformation. Overall, drug-resistant epilepsy occurs in approximately one quarter of affected individuals. When identified, genetic etiologies are very heterogeneous.

Randomized placebo-controlled crossover trial of memantine in children with epileptic encephalopathy.

Memantine is an N-methyl-D-aspartate receptor antagonist, approved for dementia treatment. There is limited evidence of memantine showing benefit for paediatric neurodevelopmental phenotypes, but no randomized placebo-controlled trials in children with developmental and epileptic encephalopathy. In this randomized double-blind placebo-controlled crossover trial (Trial registration: https://clinicaltrials.gov/ct2/show/NCT03779672), patients with developmental and epileptic encephalopathy received memantine and placebo, each for a 6-week period separated by a 2-week washout phase. Electroencephalography, seizure diary, patient caregivers' global impression, serum inflammatory markers and neuropsychological evaluation were performed at baseline and after each treatment phase. The primary outcome measure was classification as a 'responder', defined as ≥2 of: >50% seizure frequency reduction, electroencephalography improvement, caregiver clinical impression improvement or clear neuropsychological testing improvement. Thirty-one patients (13 females) enrolled. Two patients withdrew prior to initiating medication and two (twins) had to be removed from analysis. Of the remaining 27 patients, nine (33%) were classified as responders to memantine versus two (7%) in the placebo group (P < 0.02). Electroencephalography improvement was seen in eight patients on memantine compared to two on placebo (P < 0.04). Seizure improvement was observed in eight patients on memantine and two on placebo (P < 0.04). Caregivers reported overall clinical improvement in 10 patients on memantine compared to seven on placebo (not significant). Statistical analysis of neuropsychological evaluation suggested improvements in symptoms of attention-deficit hyperactivity disorder and autism. Memantine is a safe and effective treatment for children with developmental and epileptic encephalopathy, having the potential to improve both seizure control and cognitive function.

Diagnostic utility of specific abnormal EEG patterns in children for determining epilepsy phenotype and presence of structural brain abnormalities.

Objective: Estimate sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of EEG findings: centrotemporal spikes, photoparoxysmal response, asymmetric photic driving, and asymmetric sleep spindles, for epilepsy phenotype and presence of structural brain abnormalities. Methods: In this case-control study we reviewed children referred for EEG over a 4-year period, with at least one of centrotemporal spikes, photoparoxysmal response, asymmetric photic driving, or asymmetric sleep spindles. This cohort was analyzed in combination with a research database of pediatric patients with seizures. Results: Centrotemporal spikes had 100% sensitivity for childhood epilepsy with centrotemporal spikes or atypical childhood epilepsy with centrotemporal spikes, but lower specificity (70%) and PPV (58%). Photoparoxysmal response had high specificity (92%) and NPV (92%) for genetic generalized epilepsy. Asymmetric photic driving had low sensitivity for structural brain abnormalities (17%), with specificity 80%. In contrast, asymmetric sleep spindles had much higher sensitivity and specificity, 44% and 97%, respectively. Conclusions: Although centrotemporal spikes are classically associated with childhood epilepsy with centrotemporal spikes, these discharges are seen in other conditions. Photoparoxysmal response is highly indicative of a genetic generalized epilepsy, though may be seen in other epilepsy phenotypes. Relative attenuation of sleep spindles is a more reliable indicator of structural brain malformation than asymmetric photic driving. Significance: The quantitative diagnostic utility of EEG findings should be considered when incorporating these results into clinical decision-making.

The spectrum of indomethacin-responsive headaches in children and adolescents.

BACKGROUND: Headaches with marked, specific response to indomethacin occur in children, but the phenotypic spectrum of this phenomenon has not been well-studied. METHODS: We reviewed pediatric patients with headache showing ≥80% improvement with indomethacin, from seven academic medical centers. RESULTS: We included 32 pediatric patients (16 females). Mean headache onset age was 10.9 y (range 2-16 y). Headache syndromes included hemicrania continua (n = 13), paroxysmal hemicrania (n = 10), primary stabbing headache (n = 2), short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (n = 1), primary exercise headache (n = 1) and primary cough headache (n = 1). Adverse events were reported in 13, most commonly gastrointestinal symptoms, which often improved with co-administration of gastro-protective agents. CONCLUSION: Indomethacin-responsive headaches occur in children and adolescents, and include headache syndromes, such as primary cough headache, previously thought to present only in adulthood. The incidence of adverse events is high, and patients must be co-treated with a gastroprotective agent.

A retrospective analysis of memantine use in a pediatric neurology clinic.

BACKGROUND: Memantine is an N-methyl-D-aspartate receptor (NMDA-R) antagonist, approved for dementia, but also studied in pediatric autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD). METHODS: We reviewed children treated with memantine in a single-centre pediatric neurology clinic. Clinical data extracted included age, sex, weight, clinical history, reason for memantine prescription, period of treatment trial and dosage, treatment response, side effects, and concomitant medications. RESULTS: Eight patients met inclusion criteria with diagnoses including developmental and epileptic encephalopathy, focal epilepsy, ASD, ADHD. Four reported clear cognitive improvement, though two of these started other concurrent treatments at the time of memantine initiation. One of three patients with poorly-controlled epilepsy, a girl with a GRIN2A variant of uncertain significance, had a clear reduction in seizure frequency. No serious adverse events were noted. CONCLUSIONS: Memantine is generally well-tolerated in children, and may have potential benefit for a broad range of pediatric neurodevelopmental disorders.

Seizures in Sotos syndrome: Phenotyping in 49 patients.

We aimed to describe the phenotypic spectrum of seizures in Sotos syndrome, a genetic condition involving overgrowth, macrocephaly, dysmorphic features, and learning disability, in which 60%-90% have NSD1 pathogenic variants. Patients were recruited from clinics and referral from support groups. Those with seizures and a clinical diagnosis of Sotos syndrome were included. Phenotyping data were collected via structured clinical interview and chart review. Forty-nine patients were included. Twenty had NSD1 testing results available; of these, 15 (75%) had NSD1 pathogenic variants. Seizure onset age ranged from 3 months to 12 years. Staring spells (absence or focal impaired awareness seizure) were the most frequently reported semiology (33/49; 67%), followed by febrile seizures (25/49; 51%) and afebrile bilateral tonic-clonic seizures (25/49; 51%). Most patients (33/49; 67%) had multiple seizure types. The majority (33/49; 67%) had seizures controlled on a single antiseizure medication or no medication. Nine (18%) had drug-resistant epilepsy. Epilepsy syndromes included febrile seizures plus, Lennox-Gastaut syndrome, childhood absence epilepsy, and generalized tonic-clonic seizures alone. The seizure phenotype in Sotos syndrome most commonly involves staring spells, afebrile tonic-clonic seizures or febrile convulsions; however, other seizure types may occur. Seizures are typically well-controlled with medication, but drug-resistant epilepsy occurs in a minority.

Serum Levels of Valproic Acid and Carbamazepine Do Not Correlate Well with End-Organ Dysfunction.

BACKGROUND: Serum levels of anticonvulsants are commonly ordered; however, the clinical utility of these laboratory tests is unclear. Clarifying the significance of anticonvulsant drug levels is essential to allow physicians to make appropriate management decisions. We aimed to determine to what extent elevated serum levels of valproic acid (VPA) and carbamazepine (CBZ) correlate with laboratory indications of end-organ dysfunction. METHODS: We reviewed a consecutive sample of patients 0-18 years of age who, over a 2-year period, had at least one blood collection in which (1) serum [VPA] or [CBZ] was tested; and (2) at least one of the following tests was performed: alanine aminotransferase (ALT), aspartate aminotransferase (AST), platelets, white blood cells (WBC), ammonia, sodium. RESULTS: 913 and 300 blood collections met criteria for VPA and CBZ, respectively. A slight increased frequency of having any abnormal laboratory value for elevated [VPA] compared to low/normal [VPA] was observed (p = 0.02; relative risk 1.27), while there was no difference in frequency of having any abnormal lab value for CBZ, nor were there significant differences for the individual lab values. When ALT and AST were plotted against [VPA] and [CBZ], no significant correlation was observed. CONCLUSION: Serum [VPA] and [CBZ] are poor indicators of risk for drug-induced end-organ dysfunction. There are likely other, individualized risk factors that explain why certain patients develop adverse effects from these medications.

A new radiopaque embolizing agent for the treatment of endoleaks after endovascular repair: influence of contrast agent on chitosan thermogel properties.

A new injectable radiopaque embolizing agent has been developed, based on chitosan thermogelling properties. Different commercial contrast agents (Isovue®, Visipaque®, and Conray®) were associated with chitosan-ß-glycerophosphate. Their impact on gelation kinetic, mechanical properties, radiopacity, and cytotoxicity was tested to evaluate the best candidate and its feasibility for the treatment of endoleaks after endovascular aneurysm repair (EVAR). Addition of contrast agents did not prevent gelation at body temperature, but it significantly increased the viscosity of the solution before gelation, delayed gelation, and reduced the gelation rate. However, using chitosan with a high degree of deacetylation and 20 vol % contrast agent made it possible to obtain a gel with rapid gelation that was visible during X-ray based guided intervention. Hydrogels exhibit relatively low mechanical properties, which are only slightly modified by the addition of contrast agents. In vitro studies have demonstrated rapid release of contrast agents from hydrogels when immersed in a saline solution (>50% within 4 h). This is suitable for embolization, as radiopacity is required only to follow the embolization procedure, while long-term radiopacity would hamper further imaging and endoleak detection. Cytotoxicity and osmolality testing of extracts demonstrated some toxicity of products released by the gel during the first few hours, which is mainly related to their hypertonicity. After the first 24 h incubation, hydrogels released no more cytotoxic compounds, suggesting that the hydrogel rapidly becomes biocompatible. Altogether, this study suggests that the new radiopaque thermogels present interesting characteristics as embolizing agents for EVAR, although their mechanical properties require improvement.

A new injectable radiopaque chitosan-based sclerosing embolizing hydrogel for endovascular therapies.

Endovascular repair of abdominal aortic aneurysms with a stent graft is limited by the persistence or recurrence of endoleaks. These are believed to be related to the recanalization of the aneurismal sac by endothelialized neochannels, which could lead to late type I and II endoleaks. Embolization has been proposed to treat or prevent endoleaks, but presently commercialized embolizing materials have several drawbacks and do not fully prevent endoleak recurrence. A novel chitosan hydrogel that is injectable, radiopaque and contains sodium tetradecyl sulfate (STS), a well-known sclerosing agent, was developed in order to combine blood flow occlusion and endothelium ablation properties. chitosan/STS hydrogels were characterized and optimized using rheometry, scanning electron microscopy, swelling and ex vivo embolization assay. They were shown to exhibit rapid gelation and good mechanical properties, as well as sclerosing properties. Their potential for the embolization of aneurysms was subjected to preliminary in vivo evaluation in a bilateral iliac aneurysm model (three dogs) reproducing persistent endoleaks after endovascular aneurysm repair (EVAR). At 3 months no endoleak was detected in any of the three aneurysms treated with chitosan/STS hydrogels. In contrast, type I endoleaks were detected in two of the three aneurysms treated with chitosan hydrogels. Generally, chitosan/STS hydrogels have great potential as embolizing and sclerosing agents for EVAR and possibly other endovascular therapies.

Preventing bladder tumor implantation with photodynamic therapy in a rat model mimicking post-fluorescence guided transurethral resection.

PURPOSE: Fluorescence guided transurethral resection has gained acknowledgment from the urological community and it is progressively becoming more applied. It has been shown to decrease the recurrence rate of nonmuscle invasive bladder cancer due to incomplete resection due to lack of visualization. The implantation of viable tumor cells seeded during transurethral resection is another reason for recurrence. We investigated whether applying photodynamic therapy on sensitized tumor cells would decrease the amount of viable intraluminal cells and tumor cell implantation. MATERIAL AND METHODS: Two models were designed to mimic the situation after fluorescence guided transurethral resection, including partly or fully de-epithelialized bladders and circulating tumor cells loaded with protoporphyrin IX. Photodynamic therapy was performed. Controls consisted of no drug with no light, light only and drug only. Immediately after photodynamic therapy the intravesical contents were retrieved and clonogenic assays were performed on cells. Bladders were harvested 10 days after cell administration and subjected to pathological analysis. RESULTS: In the photodynamic therapy and control groups tumor volume was proportional to the instilled cell load. Clonogenic assays showed that viable cells were decreased a tenth of the initial administered amount. Tumor implantation decreased to less than a fifth of control values. CONCLUSIONS: Photodynamic therapy can effectively decrease the amount of viable tumor cells in the bladder lumen. This results in a significant decrease in tumor implantation. This technique could possibly be used to further decrease the recurrence rate of nonmuscle invasive bladder cancer.

Analysis of differential PDT effect in rat bladder tumor models according to concentrations of intravesical hexyl-aminolevulinate.

The hexylester of 5-aminolevulinic acid (HAL) is a very efficient precursor of the photosensitizer protoporphyrin IX (PpIX) for photodynamic therapy (PDT). Our previous study, performed in rat orthotopic bladder tumors, indicated an opposite effect of HAL/PpIX-PDT according to HAL concentration. The present study investigated possible reasons for this differential effect considering the impact of extracted amounts of PpIX in normal and tumor bearing bladders along with PpIX distribution in distinctive histopathological layers. High performance liquid chromatography (HPLC) analysis of tumor and normal bladder tissues after 8 mM and 16 mM HAL instillation showed that PpIX was the main porphyrin species. The PpIX production in tumor bladders instilled with 8 mM HAL was significantly higher than after 16 mM HAL. Fluorescence confocal microscopy demonstrated a punctuate bright fluorescence pattern in tumor zones of bladders instilled with 8 mM HAL, whereas a more diffuse cytoplasmatic fluorescence distribution was observed after 16 mM HAL instillation. Immunofluorescence staining together with transmission electron microscopy showed severe mitochondrial damage in tumor zones of bladders treated with 8 mM HAL/PpIX PDT, with intact mitochondria in tumor zones of bladders treated with 16 mM HAL/PpIX PDT. We conclude that the differential response to HAL/PpIX PDT in function of HAL concentrations could be attributed to diminished PpIX synthesis and differential intracellular localisation of PpIX. Mitochondria were shown to be the critical photodamaged sites of HAL/PpIX PDT and as such tissue sensitivity to treatment can be estimated through investigation of intracellular PpIX distribution.

Orthotopic animal models for oncologic photodynamic therapy and photodiagnosis.

Photodynamic therapy is a complex treatment modality where a large array of factors can influence therapeutic outcome. Vascularization, vessel permeability, oxygenation and light distribution in the tissue as well as immune response play a key role in the photodynamic process. Each of these factors can be influenced by the choice of the animal model. It is therefore of the utmost importance to choose an appropriate model for pre-clinical oncologic PDT studies. Orthotopic tumor models present the closest resemblance to the clinical situation with regard to the elements involved in PDT. We present here a brief organ specific overview of the different orthotopic animal models that can be used for in vivo photodynamic therapy studies.

A novel orthotopic bladder tumor model with predictable localization of a solitary tumor.

An ideal bladder tumor model consists in an orthotopic solitary tumor with a well-defined localization and stage, as well as unaltered normal mucosa. None of the existing models covers all these requirements. We have created a new model, suitable for diagnostic and therapeutic purposes. Female Fisher rats were divided into different groups according to bladder preconditioning and tumor cell (AY27) administration. Generalized desepithelialization was obtained by an intravesical instillation of HCl, neutralized by NaOH. Localized desepithelialization of the bladder fundus was the result of application of a micro-swab, imbibed with the same chemicals. Tumor cells administration was either generalized (intravesical instillation) or focal (micro swab). No bladder perforations were observed. Generalized desquamation always produced multifocal tumors, whereas focal application of HCl/NaOH resulted in solitary tumors of the bladder fundus, irrespective of the method of tumor cell administration. Only hyperplasia could be detected at day 3. AY27 cells were covered by umbrella cells at day 5 and subepithelial AY 27 tumor nests, covered by full thickness epithelium were observed day 7.