Design and implementation of a multisite data repository for pediatric epilepsy

This dissertation established a software-hardware integrated design for a multisite data repository in pediatric epilepsy. A total of 16 institutions formed a consortium for this web-based application. This innovative fully operational web application allows users to upload and retrieve information through a unique human-computer graphical interface that is remotely accessible to all users of the consortium. A solution based on a Linux platform with My-SQL and Personal Home Page scripts (PHP) has been selected. Research was conducted to evaluate mechanisms to electronically transfer diverse datasets from different hospitals and collect the clinical data in concert with their related functional magnetic resonance imaging (fMRI). What was unique in the approach considered is that all pertinent clinical information about patients is synthesized with input from clinical experts into 4 different forms, which were: Clinical, fMRI scoring, Image information, and Neuropsychological data entry forms. A first contribution of this dissertation was in proposing an integrated processing platform that was site and scanner independent in order to uniformly process the varied fMRI datasets and to generate comparative brain activation patterns. The data collection from the consortium complied with the IRB requirements and provides all the safeguards for security and confidentiality requirements. An 1-MR1-based software library was used to perform data processing and statistical analysis to obtain the brain activation maps. Lateralization Index (LI) of healthy control (HC) subjects in contrast to localization-related epilepsy (LRE) subjects were evaluated. Over 110 activation maps were generated, and their respective LIs were computed yielding the following groups: (a) strong right lateralization: (HC=0%, LRE=18%), (b) right lateralization: (HC=2%, LRE=10%), (c) bilateral: (HC=20%, LRE=15%), (d) left lateralization: (HC=42%, LRE=26%), e) strong left lateralization: (HC=36%, LRE=31%). Moreover, nonlinear-multidimensional decision functions were used to seek an optimal separation between typical and atypical brain activations on the basis of the demographics as well as the extent and intensity of these brain activations. The intent was not to seek the highest output measures given the inherent overlap of the data, but rather to assess which of the many dimensions were critical in the overall assessment of typical and atypical language activations with the freedom to select any number of dimensions and impose any degree of complexity in the nonlinearity of the decision space.

In vivo characterization of epileptic tissue with optical spectroscopy

For children with intractable seizures, surgical removal of epileptic foci, if identifiable and feasible, can be an effective way to reduce or eliminate seizures. The success of this type of surgery strongly hinges upon the ability to identify and demarcate those epileptic foci. The ultimate goal of this research project is to develop an effective technology for detection of unique in vivo pathophysiological characteristics of epileptic cortex and, subsequently, to use this technology to guide epilepsy surgery intraoperatively. In this PhD dissertation the feasibility of using optical spectroscopy to identify uniquein vivo pathophysiological characteristics of epileptic cortex was evaluated and proven using the data collected from children undergoing epilepsy surgery. ^ In this first in vivo human study, static diffuse reflectance and fluorescence spectra were measured from the epileptic cortex, defined by intraoperative ECoG, and its surrounding tissue from pediatric patients undergoing epilepsy surgery. When feasible, biopsy samples were taken from the investigated sites for the subsequent histological analysis. Using the histological data as the gold standard, spectral data was analyzed with statistical tools. The results of the analysis show that static diffuse reflectance spectroscopy and its combination with static fluorescence spectroscopy can be used to effectively differentiate between epileptic cortex with histopathological abnormalities and normal cortex in vivo with a high degree of accuracy. ^ To maximize the efficiency of optical spectroscopy in detecting and localizing epileptic cortex intraoperatively, the static system was upgraded to investigate histopathological abnormalities deep within the epileptic cortex, as well as to detect unique temporal pathophysiological characteristics of epileptic cortex. Detection of deep abnormalities within the epileptic cortex prompted a redesign of the fiberoptic probe. A mechanical probe holder was also designed and constructed to maintain the probe contact pressure and contact point during the time dependent measurements. The dynamic diffuse reflectance spectroscopy system was used to characterize in vivo pediatric epileptic cortex. The results of the study show that some unique wavelength dependent temporal characteristics (e.g., multiple horizontal bands in the correlation coefficient map γ(λref = 800 nm, λcomp ,t)) can be found in the time dependent recordings of diffuse reflectance spectra from epileptic cortex defined by ECoG.^

Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines

Effective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines, which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage in rat models. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain.

Outpatient Mental Health Services in Mozambique: Use and Treatments.

To describe current outpatient mental health service use and treatments in Mozambique, the authors reviewed registry entries for 2,071 outpatient psychiatric visits at the Beira Central Hospital in Sofala Province from January 2012 to September 2014. Service use was most common for schizophrenia, followed by epilepsy, delirium, and organic behavioral disorders. Only 3% of consultations for schizophrenia were first-visit patients. Treatment seeking among women was more likely for mood and neurotic disorders and less likely for substance use disorders and epilepsy. First-generation antipsychotics, most often paired with promethazine, dominated treatment regimens. Evidence-based reforms are needed to improve identification of mood disorders and broaden care beyond severe mental disorders.

BDNF-TrkB Signaling in Single-Spine Structural Plasticity

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

Functional Evaluation of Causal Mutations Identified in Human Genetic Studies

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

The Role of Dopaminergic Systems in the Neurobehavioral Teratology of Organophosphates in Zebrafish

Background: Organophosphate (OP) pesticides are well-known developmental neurotoxicants that have been linked to abnormal cognitive and behavioral endpoints through both epidemiological studies and animal models of behavioral teratology, and are implicated in the dysfunction of multiple neurotransmitters, including dopamine. Chemical similarities between OP pesticides and organophosphate flame retardants (OPFRs), a class of compounds growing in use and environmental relevance, have produced concern regarding whether developmental exposures to OPFRs and OP pesticides may share behavioral outcomes, impacts on dopaminergic systems, or both. Methods: Using the zebrafish animal model, we exposed developing fish to two OPFRs, TDCIPP and TPHP, as well as the OP pesticide chlorpyrifos, during the first 5 days following fertilization. From there, the exposed fish were assayed for behavioral abnormalities and effects on monoamine neurochemistry as both larvae and adults. An experiment conducted in parallel examined how antagonism of the dopamine system during an identical window of development could alter later life behavior in the same assays. Finally, we investigated the interaction between developmental exposure to an OPFR and acute dopamine antagonism in larval behavior. Results: Developmental exposure to all three OP compounds altered zebrafish behavior, with effects persisting into adulthood. Additionally, exposure to an OPFR decreased the behavioral response to acute D2 receptor antagonism in larvae. However, the pattern of behavioral effects diverged substantially from those seen following developmental dopamine antagonism, and the investigations into dopamine neurochemistry were too variable to be conclusive. Thus, although the results support the hypothesis that OPFRs, as with OP pesticides such as chlorpyrifos, may present a risk to normal behavioral development, we were unable to directly link these effects to any dopaminergic dysfunction.

Seizure burden and neurodevelopmental outcome in neonates with hypoxic-ischemic encephalopathy.

Aim: To examine the relationship between electrographic seizures and long-term outcome in neonates with hypoxic-ischemic encephalopathy (HIE). Method: Full-term neonates with HIE born in Cork University Maternity Hospital from 2003 to 2006 (pre-hypothermia era) and 2009 to 2012 (hypothermia era) were included in this observational study. All had early continuous electroencephalography monitoring. All electrographic seizures were annotated. The total seizure burden and hourly seizure burden were calculated. Outcome (normal/abnormal) was assessed at 24 to 48 months in surviving neonates using either the Bayley Scales of Infant and Toddler Development, Third Edition or the Griffiths Mental Development Scales; a diagnosis of cerebral palsy or epilepsy was also considered an abnormal outcome. Results: Continuous electroencephalography was recorded for a median of 57.1 hours (interquartile range 33.5-80.5h) in 47 neonates (31 males, 16 females); 29 out of 47 (62%) had electrographic seizures and 25 out of 47 (53%) had an abnormal outcome. The presence of seizures per se was not associated with abnormal outcome (p=0.126); however, the odds of an abnormal outcome increased over ninefold (odds ratio [OR] 9.56; 95% confidence interval [95% CI] 2.43-37.67) if a neonate had a total seizure burden of more than 40 minutes (p=0.001), and eightfold (OR: 8.00; 95% CI: 2.06-31.07) if a neonate had a maximum hourly seizure burden of more than 13 minutes per hour (p=0.003). Controlling for electrographic HIE grade or treatment with hypothermia did not change the direction of the relationship between seizure burden and outcome. Interpretation: In HIE, a high electrographic seizure burden is significantly associated with abnormal outcome, independent of HIE severity or treatment with hypothermia.

Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model

TORRES, F ; FILHO, M.S. ; ANTUNES, C. ; KALININE, E. ; ANTONIOLLI, E. ; PORTELA, Luis Valmor ; SOUZA, Diogo Onofre ; TORT, A. B. L. . Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model. Experimental Neurology , v. 221, p. 296-306, 2010

Mutations in SNX14 cause a distinctive autosomal-recessive cerebellar ataxia and intellectual disability syndrome

Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.

Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model

TORRES, F ; FILHO, M.S. ; ANTUNES, C. ; KALININE, E. ; ANTONIOLLI, E. ; PORTELA, Luis Valmor ; SOUZA, Diogo Onofre ; TORT, A. B. L. . Electrophysiological effects of guanosine and MK-801 in a quinolinic acid-induced seizure model. Experimental Neurology , v. 221, p. 296-306, 2010

Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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Gene-environment interactions in Autism Spectrum Disorders (ASD)

Spectrum Disorder (ASD), is a heterogeneous neurodevelopmental disorder with na estimated global prevalence rate of 17:10000, and a male to female ratio of 4:1. Patients with ASD presente language and communication difficulties and stereotyped behaviours. Comorbidity with other disorders, such as Intelectual Disability, Fragile-X syndrome (FXS) epilepsy and tuberous sclerosis frequently occurs. ASD presents amultifactorial etiopathology, and genetic factos alone are not suficiente to explain how the syndrome arises, with recente studies establishing ASD heritability at approximately 50%. Pre-, peri- and post-natal exposure to toxic environmental factos has been implicated in the development of ASD. Involvement of epigenetic regulatory mechanisms has been suggested, supported by the occurrence of autistic symptoms in patients with disorders aris ing from epigenetic mutations, such as FXS. A polygenic and epistatic model is a strong hypothesis to explain ASD. The main goal of this project is to identify specific exposure patterns to environmental toxicants in children diagnosed with ASD and integrate the results with genetic and epigenetic data.

Neuropsychiatric disease (NPD) clustering in families with Autism Spectrum Disorder (ASD): genetic, epigenetic and environmental issues

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication/interaction and by unusual repetitive and restricted behaviors and interests. ASD often co-occurs in the same families with other neuropsychiatric diseases (NPD), such as intellectual disability, schizophrenia, epilepsy, depression and attention deficit hyperactivity disorder. Genetic factors have an important role in ASD etiology. Multiple copy number variants (CNVs) and single nucleotide variants (SNVs) in candidate genes have been associated with an increased risk to develop ASD. Nevertheless, recent heritability estimates and the high genotypic and phenotypic heterogeneity characteristic of ASD indicate a role of environmental and epigenetic factors, such as long noncoding RNA (lncRNA) and microRNA (miRNA), as modulators of genetic expression and further clinical presentation. Both miRNA and lncRNA are functional RNA molecules that are transcribed from DNA but not translated into proteins, instead they act as powerful regulators of gene expression. While miRNA are small noncoding RNAs with 22-25 nucleotides in length that act at the post-transcriptional level of gene expression, the lncRNA are bigger molecules (>200 nucleotides in length) that are capped, spliced, and polyadenylated, similar to messenger RNA. Although few lncRNA were well characterized until date, there is a great evidence that they are implicated in several levels of gene expression (transcription/post-transcription/post-translation, organization of protein complexes, cell– cell signaling as well as recombination) as shown in figure 1.

Abdominal epilepsy as an unusual cause ofabdominal pain: a case report.

Introduction: Abdominal pain, in etiology sometimes difficult to be defined, is a frequent complaint in childhood. Abdominal epilepsy is a rare cause of abdominal pain. Objectives: In this article, we report on 5 year old girl patient with abdominal epilepsy. Methods: Some investigations (stool investigation, routine blood tests, ultrasonography (USG), electrocardiogram (ECHO) and electrocardiograpy (ECG), holter for 24hr.) were done to understand the origin of these complaints; but no abnormalities were found. Finally an EEG was done during an episode of abdominal pain and it was shown that there were generalized spikes especially precipitated by hyperventilation. The patient did well on valproic acid therapy and EEG was normal 1 month after beginning of the treatment. Discussion: The cause of chronic recurrent paroxymal abdominal pain is difficult for the clinicians to diagnose in childhood. A lot of disease may lead to paroxysmal gastrointestinal symptoms like familial mediterranean fever and porfiria. Abdominal epilepsy is one of the rare but easily treatable cause of abdominal pain. Conclusion: In conclusion, abdominal epilepsy should be suspected in children with recurrent abdominal pain.