Browsing by Author "Chaves, J."
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- APOE isoforms in focal epilepsies: an association study in a Portuguese populationPublication . Martins da Silva, A.; Leal, B.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Branco, R.C.; Ferreira, J.; Costa, P.; Martins da Silva, B.Purpose: Apolipoprotein E (ApoE) is the main lipoprotein secreted in brain. It has a critical immuno-modulatory function, influences neurotransmission and is involved in repairing damaged neurons. ApoE e4 allele is an isoform of ApoE with altered protein function previously associated with refractoriness and early onset epilepsy. Our purpose was to investigate if apoE isoforms are risk factors for partial epilepsy and to correlate genotypes with anti-epileptic drug response. Methods: A cohort of 230 epileptic patients with partial epilepsies from the outpatient clinic at HSA-CHP [109F, 121M; mean age = 44 13 years, age of onset = 15 13 years; 168 patients with Drug Refractory Epilepsy (DRE)] was compared with a cohort of 301 healthy individuals (HI) in a case control study. ApoE isoforms were genotype by RFLP-PCR methodology. Results: ApoE e4 allele frequency was higher in epileptic group when compared with HI (10.6% vs. 7.6%, p = n.s., OR = 1.44, 95% CI: 0.945–2.20). Anti-epileptic Drug response was not influenced by apoE isoforms. Conclusion: Our results suggest that ApoE e4 may be a risk factor for partial epilepsy development. ApoE e4 is associated with CNS network instability, with lower protection against oxidative and inflammatory cascade. These could influence neuronal growth and recovery leading to a chronic vicious cycle of damage and neuronal loss contributing to seizures development. These observations should be confirmed in a larger cohort.
- Circulating microRNAs as potential biomarkers for genetic generalized epilepsies: a three microRNA panelPublication . Martins‐Ferreira, R.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Chorão, R.; Freitas, J.; Samões, R.; Boleixa, D.; Lopes, J.; Ramalheira, J.; Silva, B.M.; Martins da Silva, A.; Costa, P. P.; Leal, B.Background and purpose: Genetic generalized epilepsies (GGEs) encompass a group of syndromes of mainly genetic causes, characterized by the involvement of both hemispheres. MicroRNAs (miRNAs) are small non-coding RNAs with a critical role in the regulation of neuronal biological processes through gene expression modulation. Dysregulated miRNA expression has been shown in epilepsy. Due to their stability in biological fluids like serum, miRNAs have assumed a prominent role in biomarker research. Our aim was to evaluate circulating levels of three miRNAs in GGE patients and assess their putative diagnostic value. Methods: MiR-146a, miR-155 and miR-132 were quantified by real-time polymerase chain reaction in the serum of 79 GGE patients (47 women, 32 men, 35.1 ± 12.4 years) and 67 healthy individuals (41 women, 26 men, 42.4 ± 10.1 years). Relative expression values were calculated using the 2-ΔΔCt method. Receiver operating characteristic curve analysis was performed to assess diagnostic value. MiRNA expression was correlated with clinicopathological features. Results: Serum levels of miR-146a and miR-155 were significantly upregulated in GGE patients relative to controls (3.13 and 6.05, respectively). Combined miR-146a, miR-155 and miR-132 serum levels performed well as a diagnostic biomarker, discriminating GGE patients from controls with an area under the curve of 0.85, 80% specificity and 73% sensitivity. Conclusions: Our results indicate that miR-146a, miR-155 and miR-132 may partake in GGE epileptogenesis. A panel of three circulating miRNAs with potential value as a GGE biomarker is reported for the first time. Novel biomarkers may help to identify new treatment targets and contribute to improved patients' quality of life through earlier diagnosis and a more precise prognosis.
- Epilepsy, hippocampal sclerosis and febrile seizures linked by common genetic variation around SCN1APublication . Kasperavičiute, D.; Catarino, C.B.; Matarin, M.; Leu, C.; Novy, J.; Tostevin, A.; Leal, B.; Hessel, E.V.S.; Hallmann, K.; Hildebrand, M.S.; Dahl, H-H.M.; Ryten, M.; Trabzuni, D.; Ramasamy, A.; Alhusaini, S.; Doherty, C.P.; Dorn, T.; Hansen, J.; Krämer, G.; Steinhoff, B.J.; Zumsteg, D.; Duncan, S.; Kälviäinen, R.K.; Eriksson, K.J.; Kantanen, A-M; Pandolfo, M.; Gruber-Sedlmayr, U.; Schlachter, K.; Reinthaler, E.M.; Stogmann, E.; Zimprich, F.; Theatre, E.; Smith, C.; Obrien, T.J.; Tan, K.M.; Petrovski, S.; Robbiano, A.; Paravidino, R.; Zara, F.; Striano, P.; Sperling, M.R.; Buono, R.J.; Hakonarson, H.; Chaves, J.; Costa, P.P.; Silva, B.M.; Da Silva, A.M.; De Graan, P.N.E.; Koeleman, B.P.C.; Becker, A.; Schoch, S.; Von Lehe, M.; Reif, P.S.; Rosenow, F.; Becker, F.; Weber, Y.; Lerche, H.; Roessler, K.; Buchfelder, M.; Hamer, H.M.; Kobow, K.; Coras, R.; Blumcke, I.; Scheffer, I.E.; Berkovic, S.F.; Weale, M.E.; Delanty, N.; Depondt, C.; Cavalleri, G.L.; Kunz, W.S.; Sisodiya, S.M.Epilepsy comprises several syndromes, amongst the most common being mesial temporal lobe epilepsy with hippocampal sclerosis. Seizures in mesial temporal lobe epilepsy with hippocampal sclerosis are typically drug-resistant, and mesial temporal lobe epilepsy with hippocampal sclerosis is frequently associated with important co-morbidities, mandating the search for better understanding and treatment. The cause of mesial temporal lobe epilepsy with hippocampal sclerosis is unknown, but there is an association with childhood febrile seizures. Several rarer epilepsies featuring febrile seizures are caused by mutations in SCN1A, which encodes a brain-expressed sodium channel subunit targeted by many anti-epileptic drugs. We undertook a genome-wide association study in 1018 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 7552 control subjects, with validation in an independent sample set comprising 959 people with mesial temporal lobe epilepsy with hippocampal sclerosis and 3591 control subjects. To dissect out variants related to a history of febrile seizures, we tested cases with mesial temporal lobe epilepsy with hippocampal sclerosis with (overall n = 757) and without (overall n = 803) a history of febrile seizures. Meta-analysis revealed a genome-wide significant association for mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures at the sodium channel gene cluster on chromosome 2q24.3 [rs7587026, within an intron of the SCN1A gene, P = 3.36 × 10(-9), odds ratio (A) = 1.42, 95% confidence interval: 1.26-1.59]. In a cohort of 172 individuals with febrile seizures, who did not develop epilepsy during prospective follow-up to age 13 years, and 6456 controls, no association was found for rs7587026 and febrile seizures. These findings suggest SCN1A involvement in a common epilepsy syndrome, give new direction to biological understanding of mesial temporal lobe epilepsy with hippocampal sclerosis with febrile seizures, and open avenues for investigation of prognostic factors and possible prevention of epilepsy in some children with febrile seizures.
- Expression of adenosine kinase in human mesial temporal lobe epilepsy with hippocampal sclerosis: A preliminary studyPublication . Leal, B.; Rangel, R.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Zenatti, L.; Santos, A.; Magalhães, T.; Martins da Silva, A.; Correia de Sá, P.; Martins da Silva, B.; Costa, P.P.Background: Adenosine is a ubiquitous homeostatic molecule that acts as an “endogenous neuromodulator”. Adenosine attenuates neuronal activity either presynaptically by inhibiting neurotransmitter release or by controlling neurotransmitter responsiveness at post-synaptic sites. Unbalanced adenosine metabolism has been implicated in pathological conditions such as epilepsy. Adenosine kinase (ADK), synthetized by astrocytes, is the key regulator of extracellular adenosine levels in the brain. Evidences from experimental studies support a role for ADK in brain injury associated with astrogliosis, a morphological hallmark of Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS). In fact, expression of astrocytic ADK was found to be increased in the hippocampus and temporal cortex of MTLE-HS patients. Overexpression of ADK decreases extracellular adenosine and consequently may cause seizures. The aim of this study was to characterize ADK gene expression in MTLE-HS patients. Methods: Previous studies used immunohistochemistry and Western blot analysis to investigate ADK expression. Here we quantified the expression levels of ADK by Real-Time PCR in the hippocampus (lesional and peri-lesional cortical area) of 10 MTLE-HS patients submitted to surgery as compared with 9 autopsy controls with no history of neurological disorders. Results: Our results showed that ADK expression levels were similar in the hippocampus and temporal cortex of MTLE-HS patients when compared to healthy controls. Conclusion: Our preliminary data demonstrate that ADK expression levels are not altered in MTLE-HS. These results do not preclude post-transcriptional ADK abnormalities at both protein and functional levels. Our results should be confirmed in a larger cohort as well as with complementary methodologies.
- Expression of miR146-a, an inflammation-associated microRNA, in Mesial Temporal Lobe EpilepsyPublication . Leal, B.; Carvalho, C.; Chaves, J.; Bettencourt, A.; Freitas, J.; Lopes, J.; Ramalheira, J.; Martins da Silva, A.; Costa, P. P.; Martins da Silva, B.Background: Neuroinflammation appears as an important epileptogenic mechanism. MicroRNAs (miRNA) are small non-coding RNA molecules that function as post-transcriptional regulators of gene expression. MicroRNas control different biological process including immune system homeostasis and function. Several evidences, both in patients and animal studies, have demonstrated an abnormal brain expression of miR-146a in Mesial Temporal Lobe Epilepsy. Knowing that miR expression is very stable in biological fluids such as plasma or serum our aim was to characterize miR146a expression in serum of MTLE patients. Methods: Expression levels of miR146a and U6B small nuclear RNA gene (reference gene) were quantified by Real-Time PCR in serum of 14 MTLE patients all with Hippocampal Sclerosis (6F, 8M, mean age= 44.1±11.7 years, age of onset= 13.5±10.6 years, 7 with Febrile Seizures antecedents). A group of 10 healthy individuals was used as control. Relative expression values were calculated using the 2-ΔΔCt method. Results: We observed that expression of miR146a was 2 fold higher in MTLE-HS patients than in controls. Conclusion: The results obtained in serum are in accordance with the results obtained from brain tissue of epileptic patients. This may confirm that miR-146a is a suitable biomarker of epileptogenesis. Additionally, it is thought that miR-146a has a role in fine-tuning the response to cytokines during epileptogenesis. Nevertheless its importance in epilepsy development it is yet not fully understood. The comprehension of this role may be relevant for the development of new therapeutic strategies.
- Human herpes virus 6B and mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS): is there a link?Publication . Leal, B.; Castelo Branco, R.; Rangel, R.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Honavar, M.; Melo Pires, M.; Santos, A.; Magalhães, T.; Lopes, J.; Ramalheira, J.; Martins da Silva, A.; Costa, P.P.; Martins da Silva, B.Purpose: Human Herpesvirus 6 (HHV-6) is a ubiquitous virus acquired mainly during the first 2 years of life. Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS) is the most frequent pharmacoresistant epilepsy. One of the most common antecedents of MTLE-HS is febrile seizures (FS). Although the aetiology of MTLE-HS remains unclear, evidences suggest that HHV-6 infection could be implicated. The objective of this study was to investigate the presence of HHV-6B DNA in the hippocampus and adjoining temporal cortex of MTLE-HS patients submitted to surgery. Methods: A total of 22 MTLE-HS (13 females and nine males) cases were studied. The mean age at surgery was 39 9 years and mean age at onset of seizures was 10 6 years. These study cohort was compared to a group of 10 epileptic patients without MTLE-HS (six females, four males; mean age = 26 15 years) and with autopsy material from nine individuals without neurological disease. HHV-6B DNA was identified by real-time PCR with specific TaqMan probes. Results: We detected HHV-6B DNA in only one hippocampus from a MTLE-HS patient. This patient had a disease duration of 36 years and a history of febrile seizures in childhood. None of the non-MTLE or controls specimens showed positivity for HHV-6B. Conclusions: Our findings do not support a relevant etiologic role for HHV-6B in MTLE-HS, at least in this population. However, the possible role of viral infection in MTLE-HS epileptogenic process, in individual cases, cannot be excluded.
- Is serotonin receptor HTR1B implicated in mesial temporal lobe epilepsy development?Publication . Leal, B.; Barreira, A.; Chaves, J.; Carvalho, C.; Bettencourt, A.; Martins da Silva, A.; Costa, P.P.; Martins da Silva, B.Background: Evidences from animal models have demonstrated that depletion of brain serotonin (5-HT), a neurotransmitter with a pivotal role in neurodevelopment and brain plasticity, lowers the threshold to induced seizures. It was also demonstrated that anti-epileptic drugs increase endogenous 5-HT concentrations and that 5HT-1B receptors could have an anticonvulsant role. Association studies have demonstrated that a polymorphism (rs6296) in 5HTR-1B gene may be a susceptibility factor for with temporal lobe epilepsy (TLE) development. The rs6296 G allele has been associated with decreased receptor activity. Our aim was to analyse the association between rs6296 and the development and clinical features of Mesial Temporal Lobe Epilepsy (MTLE) in a Portuguese population. Material and methods: A cohort of 121 MTLE patients (67 F, 54 M, mean age = 44 ± 11 years, age of onset = 13 ± 9 years) was compared with a cohort of 192 healthy individuals (HI). All patients had Hippocampal Sclerosis (MTLE–HS). Genotyping was performed by TaqMan real time PCR methodology. Results: rs6296 G allele was overrepresented in MTLE patients compared to controls (80.2% in MTLE vs 72.1% in HI, p = 0.029 OR = 1.561 (1.060–2.298)). We constituted 2 MTLE–HS sub-groups, according to febrile seizure antecedents and no differences in rs6596 allelic or genotypic frequencies were found. Conclusion: The rs6296 G allele may be a susceptibility factor to MTLE–HS development. Since these receptors have an anticonvulsant role, a reduction in their activity could lower the threshold for seizure development.
- Lack of Cystatin B Protein as a Cause Of Myoclonic EpilepsyPublication . Amaral, O.; Duarte, A.; Pinto, E.; Freitas, J.; Chaves, J.Unverricht-Lundborg disease (ULD; MIM 254800) is the most frequent cause of progressive myoclonic epilepsy. CSTB mutations (locus 21q22.3; MIM 601145), with cystatin B loss of function and subsequent loss of lysosomal association, have been described as the major cause of this disease.
- Mesial temporal lobe epilepsy and serotonin: the role of HTR2A receptorPublication . Chaves, J.; Leal, B.; Carvalho, C.; Bettencourt, A.; Bras, S.; Barreira, A.; Boleixa, D.; Martins da Silva, A.; Costa, P.P.; Martins da Silva, B.Purpose: Evidences from animal models have demonstrated that depletion of brain serotonin (5-HT), a neurotransmitter with a pivotal role in neurodevelopment and brain plasticity, lowers the threshold to induced seizures. It was also demonstrated that anti-epileptic drugs increase endogenous 5-HT concentrations. Studies in brain tissue from Mesial Temporal lobe Epilepsy (MTLE) patients have showed that serotonin type 2a receptor (HTR2A) is downregulated in these patients. HTR2A expression levels may be modulated by a 102 T>C polymorphism. The aim of this study was to analyse the association between 102T>C polymorphism and the development and clinical features of MTLE-HS in a Portuguese population. Methods: A cohort of 112 MTLE-HS patients (62F, 50M, mean age = 44 11 years, age of onset = 13 9 years, 97 patients with drug refractory epilepsy) was compared with a cohort of 183 healthy individuals (HI). Genotyping was performed by Real Time PCR using High Melting Resolution methodology. Results: HTR2A 102 T>C genotype frequencies were similar between patients and controls (TT: 24.1% vs. 25.1% in HI; TC: 45.5% vs. 43.2% in HI; CC: 31.3% vs. 31.7% in HI). No association was found between this polymorphism and MTLE-HS clinical features (age of onset, FS antecedents and anti-epileptic drug response). Conclusion: The present results do not provide evidence that HTR2A polymorphism 102T>C may confer susceptibility to MTLE-HS. Nevertheless a possible role for the serotonergic system in epileptogenesis cannot be excluded. The study of other 5-HT receptors and transporters is underway.
- Unverricht-Lundborg disease: development of splicing therapeutic approaches for a patient with an homozygous mutation in the cystatin B genePublication . Matos, L.; Duarte, A.J.; Ribeiro, D.; Jordan, P.; Prata, M.J.; Chaves, J.; Desviat, L.R.; Pérez, B.; Amaral, O.; Alves, S.Unverricht-Lundborg disease (ULD) is the most common form of progressive myoclonic epilepsy worldwide. It is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion, missense, nonsense, frameshift and mutations that may lead to alternative splicing have been described as causal of ULD. Recently, our group described an ULD patient who is homozygous for a new synonymous mutation (c.66G>A; p.Q22Q) located at the last nucleotide of exon 1. The transcriptional profile analysis allowed the identification of two CSTB splice variants, one of normal size with the G>A change and other with partial inclusion of intron 1 due to activation of a cryptic splice-site inside the intronic sequence. To correct the splice defect, here we developed antisense oligonucleotide and U1snRNA mediated therapeutic strategies. U1 is required for splice donor site (SDS) recognition of pre-mRNAs and initiates the splicing process. The mutation c.66G>A interferes with the recognition of the SDS by U1. In a first approach, to reduce missplicing we generated four U1 construct isoforms with increasing complementarity to the SDS. Transfection of patient-derived fibroblasts with different concentrations of the adapted U1 vectors did not allowed the correction of the aberrant transcript. In a second strategy, we have designed a specific lock nucleic-acid (LNA) oligonucleotide to block the activated cryptic splice-site in intron 1. Normal splicing pattern of a single transcript with the synonymous change G>A was successfully rescued after LNA transfection in patient cells. The therapeutic effect showed to be dose-dependent. These results suggest that antisense therapy might be a potential alternative or adjunct treatment strategy for patients holding splicing changes in CSTB gene. As far as we know this is the first report of a patient tailored therapy in cells of an ULD patient.