Is autopsy tissue a valid control for epilepsy surgery tissue in microRNA studies?

MicroRNAs (miRNAs) are differentially expressed in the brain under pathologic conditions and may therefore represent both therapeutic targets and diagnostic or prognostic biomarkers for neurologic diseases, including epilepsy. In fact, miRNA expression profiles have been investigated in the hippocampi of patients with epilepsy in comparison with control, nonepileptic cases. Unfortunately, the interpretation of these data is difficult because surgically resected epileptic tissue is generally compared with control tissue obtained from autopsies. To challenge the validity of this approach, we performed an miRNA microarray on the laser microdissected granule cell layer of the human hippocampus obtained from surgical samples of patients with epilepsy, autoptic nonepileptic controls, and patients with autoptic epilepsy, using the latter as internal control. Unfortunately, it is extremely difficult to collect autopsy material from documented epilepsy individuals who died of non–epilepsy-related causes—we found only two such cases. However, hierarchical clustering of all samples showed that those obtained from autopsies of patients with epilepsy segregated with the other autoptic samples (controls) and not with the bioptic tissues from the surgery patients, suggesting that the origin of the tissue (surgery or autopsy) may be prevalent over the underlying pathology (epilepsy or not epilepsy). Even taking into account the limitations due to the small number of cases, this observation arises concerns on the use of autopsy tissue as control for this kind of studies

Discovery of novel mutations in the dihydropyrimidine dehydrogenase gene associated with toxicity of fluoropyrimidines and viewpoint on preemptive pharmacogenetic screening in patients

Background: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the metabolic pathway of 5-fluorouracil (5-FU) and other fluoropyrimidines to inactive compounds. For this reason, severe, life-threatening toxicities may occur in patients with deficient DPD activity when administered standard doses of 5-FU and its prodrugs. Materials and methods: We selected three patients with colorectal adenocarcinoma who displayed unexpected severe adverse reactions after treatment with 5-FU and capecitabine. To investigate the possible involvement of deficient variants of the DPD gene (DPYD), a denaturing HPLC (dHPLC) approach followed by target exon sequencing of DPYD was performed on DNA extracted from peripheral blood. Results: Three novel non-synonymous mutations of DPYD, c.2509-2510insC, c.1801G>C, and c.680G>A, were detected in these subjects. Due to the absence of other deficient variants of DPYD and the compatibility of adverse reactions with fluoropyrimidine treatment, the novel variants were associated with a poor-metabolizer phenotype. Conclusions: Stratification of patients on the basis of their genotype may help prevent toxicity, and the large body of evidence about the pathogenesis of fluoropyrimidine-induced adverse reactions strongly encourages the adoption of best practice recommendations to appropriately address this important clinical issue. This approach is of utmost importance within a preventive, prognostic, and personalized approach to patient care in the oncology setting

Effects of cardiac resynchronization therapy on systemic inflammation and neurohormonal pathways in heart failure

Background: The effect of cardiac resynchronization therapy (CRT) on systemic inflammation and neurohormonal alterations associated with heart failure is not well characterized. Accordingly, we aimed to assess the long term effects of CRT on systemic inflammation and neurohormonal factors in heart failure patients. Methods and results: In 47 HF patients (NYHA III–IV) we evaluated, at baseline and after one year of CRT: TNF-α, TNF soluble receptors (sTNFR1 and sTNFR2), insulin-like growth factor-1α (IGF-1α), adiponectin, norepinephrine, pro-atrial natriuretic peptide (pro-ANP), N-terminal-pro-brain natriuretic peptide (NT-proBNP) and angiotensin II, NYHA functional class, quality of life (the Minnesota Living with Heart Failure questionnaire), a 6-minute walk test and an echocardiogram. Long-term CRT decreased activation of renin–angiotensin system (RAS) only in patients with reverse remodelling. It failed to prevent a decline in adiponectin levels, regardless of reverse remodelling. NT-proBNP remained unchanged in patients with reverse remodelling, whereas its levels increased in those without reverse remodelling. IGF-1α increased with CRT, whereas CRT had no effect on pro-ANP and inflammatory markers. Conclusions: Long-term CRT is associated with decreased RAS activation and stabilization of NT-proBNP in heart failure patients with reverse remodelling. Long-term CRT, with or without reverse remodelling, does not affect systemic inflammation and fails to prevent a decline in adiponectin

MicroRNA profiles in hippocampal granule cells and plasma of rats with pilocarpine-induced epilepsy - Comparison with human epileptic samples

The identification of biomarkers of the transformation of normal to epileptic tissue would help to stratify patients at risk of epilepsy following brain injury, and inform new treatment strategies. MicroRNAs (miRNAs) are an attractive option in this direction. In this study, miRNA microarrays were performed on laser-microdissected hippocampal granule cell layer (GCL) and on plasma, at different time points in the development of pilocarpine-induced epilepsy in the rat: latency, first spontaneous seizure and chronic epileptic phase. Sixty-three miRNAs were differentially expressed in the GCL when considering all time points. Three main clusters were identified that separated the control and chronic phase groups from the latency group and from the first spontaneous seizure group. MiRNAs from rats in the chronic phase were compared to those obtained from the laser-microdissected GCL of epileptic patients, identifying several miRNAs (miR-21-5p, miR-23a-5p, miR-146a-5p and miR- 181c-5p) that were up-regulated in both human and rat epileptic tissue. Analysis of plasma samples revealed different levels between control and pilocarpine-treated animals for 27 miRNAs. Two main clusters were identified that segregated controls from all other groups. Those miRNAs that are altered in plasma before the first spontaneous seizure, like miR-9a-3p, may be proposed as putative biomarkers of epileptogenesis

Treatment with metformin in twelve patients with Lafora disease

Background: Lafora disease (LD) is a rare, lethal, progressive myoclonus epilepsy for which no targeted therapy is currently available. Studies on a mouse model of LD showed a good response to metformin, a drug with a well known neuroprotective effect. For this reason, in 2016, the European Medicines Agency granted orphan designation to metformin for the treatment of LD. However, no clinical data is available thus far. Methods: We retrospectively collected data on LD patients treated with metformin referred to three Italian epilepsy centres. Results: Twelve patients with genetically confirmed LD (6 EPM2A, 6 NHLRC1) at middle/late stages of disease were treated with add-on metformin for a mean period of 18 months (range: 6-36). Metformin was titrated to a mean maintenance dose of 1167 mg/day (range: 500-2000 mg). In four patients dosing was limited by gastrointestinal side-effects. No serious adverse events occurred. Three patients had a clinical response, which was temporary in two, characterized by a reduction of seizure frequency and global clinical improvement. Conclusions: Metformin was overall safe in our small cohort of LD patients. Even though the clinical outcome was poor, this may be related to the advanced stage of disease in our cases and we cannot exclude a role of metformin in slowing down LD progression. Therefore, on the grounds of the preclinical data, we believe that treatment with metformin may be attempted as early as possible in the course of LD

Progressive myoclonus epilepsies due to SEMA6B mutations. New variants and appraisal of published phenotypes

Variants of SEMA6B have been identified in an increasing number of patients, often presenting with progressive myoclonus epilepsy (PME), and to lesser extent developmental encephalopathy, with or without epilepsy. The exon 17 is mainly involved, with truncating mutations causing the production of aberrant proteins with toxic gain of function. Herein, we describe three adjunctive patients carrying de novo truncating SEMA6B variants in this exon (c.1976delC and c.2086C > T novel; c.1978delC previously reported). These subjects presented with PME preceded by developmental delay, motor and cognitive impairment, worsening myoclonus, and epilepsy with polymorphic features, including focal to bilateral seizures in two, and non-convulsive status epilepticus in one. The evidence of developmental delay in these cases suggests their inclusion in the “PME plus developmental delay” nosological group. This work further expands our knowledge of SEMA6B variants causing PMEs. However, the data to date available confirms that phenotypic features do not correlate with the type or location of variants, aspects that need to be further clarified by future studie

The Collaborative for the Research on Black Women and Girls

<p>Seven patients per group. **P<0.01 Mann-Whitney U test. Representative granule cell layer hippocampal sections from patients without granule cell pathology (B) or with type-2 GCP (C) exhibiting DAB-labeled ANTXR1-like immunoreactivity (LI). Omitting the primary antibody to estimate nonspecific signal yielded completely negative labeling (data not shown). Note a widespread increase in ANTXR1-LI in granule cells from patients with type-2 GCP (C).</p

The 5th International Lafora Epilepsy Workshop: Basic science elucidating therapeutic options and preparing for therapies in the clinic

Lafora disease (LD) is both a fatal childhood epilepsy and a glycogen storage disease caused by recessive mutations in either the Epilepsy progressive myoclonus 2A (EPM2A) or EPM2B genes. Hallmarks of LD are aberrant, cytoplasmic carbohydrate aggregates called Lafora bodies (LBs) that are a disease driver. The 5th International Lafora Epilepsy Workshop was recently held in Alcala de Henares, Spain. The workshop brought together nearly 100 clinicians, academic and industry scientists, trainees, National Institutes of Health (NIH) representation, and friends and family members of patients with LD. The workshop covered aspects of LD ranging from defining basic scientific mechanisms to elucidating a LD therapy or cure and a recently launched LD natural history study

Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.Peer reviewe