Browsing by Author "Martins da Silva, A."
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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.
- 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.
- How genetic characterization of Narcolepsy and hypersomnia is useful on phenotype definitionPublication . Martins da Silva, A.; Lopes, J.; Ramalheira, J.; Carvalho, C.; Costa, P.P.; Silva, B.M.Introduction. The determination of HLA class II genotype is widely used to confirm the diagnosis of narcolepsy with or without cataplexy. It is known from studies carried out in the 80’s and 90’s that genetic markers, particularly HLA-DR2 (HLA-DRB1*15) and later DQB1*06:02, are strongly associated with susceptibility to narcolepsy (N). First studies in 1984 showed values of 100% of positive HLA-DR2 (Langdon et al; Lancet 1984) in narcoleptic patients with cataplexy (NC). Mignot et al (Sleep 1997) found values of 76.1% of HLA-DQB1*06:02 positive in NC and 40.9% in N. More recent studies emphasize difference between children and adults for HLA-DQB1*06:02. Values of 93.7% (adults) vs 92.6% (children) in NC and a frequency of 78,6% in adults vs 52.9% in children were found in N (Nevsimalova et al; J Neurol 2013). The dissemination of HLA genotyping was the result of two convergent reasons: i) the method is reliable, easy to perform and reassures the clinician; ii) the assay is less invasive than other methodologies, namely those involving cerebrospinal fluid (CSF) samples. Another contributing factor is the wide acceptance of the hypothesis of an autoimmune origin for Narcolepsy (a clinical field in which the relevance of HLA system is generally accepted). This hypothesis finds support in the virtually absent levels of hypocretin peptides in the CSF of patients with NC, which is postulated to be due to the autoimmune destruction of hypocretin producing neurons (Burgess et al; J Neurosci 2012). Aims. To evaluate the contribution of genetic markers (HLA) to the differential diagnosis between narcolepsy with (NC) or without cataplexy (N) and hypersomnia (H) and their relevance in the context of our population (Northern Portugal). Patients and methods. A cohort of 113 patients with sleep and hypersomnia complaints were observed at the Outpatient Sleep Clinic from Hospital Santo Antonio/CH Porto and were assessed by clinical, night sleep polygraphic recording, MSLT on the following day, blood sampling in a routine method. Data from laboratory parameters was confronted with the clinical diagnostic hypothesis. Clinical reevaluation of the patients was considered if results did not match. Of these patients, classified as NC, N or H (according to ICSD2, 2005), 38 were NC (age at testing: mean, 32.8 years; median, 30 years); 13 N (age at testing: mean, 34.2 years; median: 36 years); 62 patients had H (age at testing: mean, 38.2 years; median, 40 years). We used a control population (CP) of 206 reportedly healthy individuals from the same geographic origin. The allele frequencies of the control population were confirmed and compared with a larger cohort of another population (2500 individuals) from the central and south regions of the country. Results. The frequency of HLA-DQB1*06:02 allele was overrepresented in N and NC patients (46% and 71% respectively), as expected, and the p value is extremely significant for NC (p < 0.0000). HLA-DQB1*02 frequency was also increased in the population with H when compared with the CP (55% vs 34%; p = 0.00396). Interestingly the frequency of the HLADQB1*03 allele was decreased in the NC vs CP group (34% vs 56%; p =0.012153). No differences were found in the HLA-DQB1*06:03 frequency between the cohort of patients and the control population. Conclusions. The HLA-DQB1*06:02 allele, a susceptibility factor to other autoimmune disorders (e.g.: MS, SLE, sarcoidosis, sclerosing cholangitis), was confirmed as a susceptibility allele also to NC in our population. The frequency of this allele in our NC patients (71%) is within the range of other studies. This value is lower when compared to studies concerning only patients with severe cataplexy (frequencies between 85-95%). Some of the differences could be due to phenotypic uncertainty or to the clinical picture evolution in different age groups (Nevsimalova et al; J Neurol 2013). Also a modification of hypocretin levels, by circadian or other oscillations and the influence of environmental factors (infections, head trauma, immunization) can be involved. The role of the potential regeneration of CNS tissue is also a subject to be explored. Given these uncertainties, genetic characterization has the potential to enhance the ability to carry out differential diagnosis among diverse excessive daytime sleepiness phenotypes, helping in the distinction of diverse entities corresponding to fundamentally different biological processes. Finally a matter to be explored is the role of HLADQB1*06:03 allele, considered by some authors as a protective factor to NC (Hor et al; Nat Genet 2010) (Van der Heide et al; Sleep 2012). Our study did not confirm this assumption.
- 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.
- 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.
- MiR-134 serum expression in Mesial Temporal Lobe Epilepsy patientsPublication . Leal, B.; Rodrigues, D.; Carvalho, C.; Ferreira, R.; Chaves, J.M.M.; Bettencourt, A.; Freitas, J.; Lopes, J. M.C.F.; Ramalheira, J.E.D.P.; Martins da Silva, A.; Costa, P.P.; Martins da Silva, B.Background and aims: Several experimental and clinical studies have suggested that microRNAs (miRNAs) could be potential epilepsy biomarkers. Nowadays, research has been focused in miR-134, a brain-specific miRNA that plays important roles in dendritic spine development and neuronal structure regulation. An upregulation of miR-134 has been reported both in brain tissue of experimental models (Jimenez-Mateos 2012) and plasma from epileptic patients (Sun 2017). It has also been observed that some anti-seizure drugs down regulate mir-134 plasmatic levels (Sun 2017) highlighting the role of this miRNA in epileptogenesis. Our aim was to quantify miR-134 serum levels in a cohort of Mesial Temporal Lobe Epilepsy (MTLE) patients and correlate with clinical characteristics such as drug response.
- Predictive power of HLA-DQB1 to identify narcoleptic patientsPublication . Silva, M.; Lopes, J.; Carvalho, C.; Cunha, D.; Costa, P.P.; Martins da Silva, A.Introduction The determination of HLA class II genotype is widely used to confirm the diagnosis of Narcolepsy without (N) or with Cataplexy (NC). The use of HLA genotyping in clinical diagnosis is reliable and is a contributing factor to the reinforcement of the wide acceptance of the hypothesis of autoimmune origin for Narcolepsy. We evaluate the contribution of genetic markers (HLA) in the differential diagnosis between narcolepsy with and without Cataplexy and their relevance in the context of our population (Northern Portugal). Materials and methods A cohort of 53 patients with Narcolepsy with Cataplexy (NC) or without (N) was studied. Patients followed up to the Outpatient Sleep Clinic of Hospital Santo António/CH Porto were assessed by clinical, night sleep polygraphic recording, MSLT on the following day. Blood sampling for HLA-DQB1∗ analysis was performed after informed consent. The genotyping was achieved by using a PCR-Sequence Specific Primer (SSP) methodology. Control Population (CP) comprised 206 reportedly healthy individuals from the same geographic origin. Data from laboratory parameters was confronted with the clinical diagnostic hypothesis. Patients’ clinical reevaluation was considered if phenotype-genotype did not match. Results Of the 53 patients, 14 were classified as N and 39 as NC. The frequency of HLA-DQB1∗06:02 allele was overrepresented in N and NC patients (43% and 69%, respectively) when compared with the control population (16%) – p value = 1.38 × 10–12 for NC. Interestingly the frequency of the HLA- DQB1∗03 allele was decreased in NC patients (36% NC vs. 56% CP, p = 0.01917). No differences were found in other DQB1∗06 frequencies between the cohort of patients and the control population. Conclusion The HLA-DQB1∗06:02 allele, a susceptibility factor for other autoimmune disorders, was confirmed as the most important susceptibility allele to NC in our population. The frequency of this allele in our NC patients (69%) is within the range of other studies. Such values pointed out to 11.8 OR for NC comparing with 3.9 OR for N patients. These findings point out to the relevance of the allele DQB1∗0602 in this sleep entity and on the autoimmune involvement in the picture of Narcolepsy–Cataplexy.