Aplicaciones de la transformada wavelet en el procesamiento de biopotenciales electrooculográficos

Conferencia impartida por el Pr. Dr. Rodolfo García Bermúdez en la ETSI Telecomunicación y ETSI Informática dentro del programa de conferencias científicas incluido en el Plan Propio.Organiza el Departamento de Tecnología ElectrónicaA partir de una panorámica general acerca de los biopotenciales electro-oculográficos, con énfasis en movimientos oculares sacádicos, se describirán algunos de los retos presentes en su procesamiento: la simulación de estas señales por medio de funciones, algunas técnicas para la eliminación de ruido y la obtención de perfiles de velocidad por medio de diferenciación aplicando transformada wavelet.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Evaluation of central and peripheral vestibular patients with the video-head impulse test

Dizziness and vertigo are highly prevalent symptoms that accompany a wide variety of conditions including peripheral vestibular dysfunction, central (vestibular) lesions and somatoform disorders. A correct diagnosis is the prerequisite for successful treatment, which should be directed towards the underlying pathophysiology. Neurophysiological methods that test the integrity of the peripheral and central vestibular system circuitry are essential to make an accurate diagnosis in clinical practice. Currently, that assessment is achieved primarily through eye movement analysis in response to semicircular canal stimulation, namely through caloric stimulation and head impulses. The quantification of the vestibulo-ocular reflex (VOR) dynamic parameters and the characterization of quick eye movements (QEM) triggered with head impulses can now be non-invasively and easily assessed with the video headimpulse test (vHIT). This provided a unique opportunity to carry out neurophysiological studies on the oculomotor responses generated by head impulses in humans. Our aim was to determine if the involvement of central vestibular pathways caused differential disturbances in VOR dynamic changes when explored with the vHIT, which could contribute not only to the differential diagnosis of patients but also to the understanding of VOR control mechanisms. We explored normal subjects and patients diagnosed with acute vestibular syndrome with spontaneous nystagmus of peripheral and central origin and hereditary neurodegenerative disorders. Looking for a simple sign of peripheral disease with the vHIT we noticed anti-compensatory eye movements (AQEM) in patients with peripheral aetiologies of spontaneous nystagmus (SN). In the first study we looked for the accuracy of AQEM to differentiate central from peripheral origins of SN. We recorded the eye movements in response to horizontal head impulses in a group of 43 consecutive patients with acute vestibular syndrome (12 with central, 31 with peripheral disorders), 5 patients after acute vestibular neurectomy (positive controls) and 39 healthy subjects (negative controls). AQEM were defined as quick eye movements (peak velocity above 50°/s) in the direction of the head movement. All patients with peripheral disorders and positive controls had AQEM (latency 231±53ms, amplitude 3.4±1.4º, velocity 166±55º/s) when their head was moved to the opposite side of the lesion. Central patients did not have AQEM. AQEM occurrence rate was higher in peripheral patients with contralesional (74±4%, mean±SD) in comparison to ipsilesional (1±4%) impulses (p<0.001). Overall diagnostic accuracy for differentiating central from peripheral patients was 96% (95% CI for AUC ROC curve: 0.90 to 1.0) for VOR gain and 100% (95% CI: 1.0 to 1.0) for AQEM occurrence rate. These results suggest that AQEM are a sign of vestibular imbalance in a peripheral deficit and should be added to VOR gain analysis in acute vestibular syndrome patients. In the second study on acute vestibular syndrome we reported on a patient with benign paroxysmal positional vertigo (BPPV) and spontaneous nystagmus due to otoconia causing a plug in the horizontal semicircular canal. The video head-impulse test revealed an eye velocity saturation with ipsilesional head impulses that normalized after liberatory maneuvers, documenting for the first time a reversible deficiency of the cupularendolymph high-frequency system dynamics. Furthermore cervical and ocular vestibular myogenic potentials were absent during stimulation of the affected side before the liberatory maneuvers, but normalized within 30 to 80 days. These observations challenge the common belief that VEMPs are evoked by otolith stimulation only, as the assumption of a reversible canal dysfunction by a plug offers a more plausible explanation for all effects. Finally, we reported on a patient presenting with a one-year history of progressive unsteadiness, particularly when in darkness. The video-Head Impulse Test (vHIT)1 (Figure 1 B) revealed a significantly reduced vestibuloocular reflex (VOR) gain in both horizontal (0.38±0.07 and 0.29±0.05) and posterior canals (0.49±0.05 and 0.38±0.06) with covert and overt corrective saccades, but normal VOR responses in both anterior canals (0.89±0.08 and 1.04±0.15), for right and left impulses, respectively. No plausible combination of end-organ lesion should be responsible for these observations. A brain MRI disclosed a left inferior cerebellar peduncle lesion suggestive of a glioma. To the best of our knowledge, this is the first report where three-dimensional vHIT, by means of peripheral-unlikely combinations of VOR lesion, has shown to be of topodiagnostic value. In the second set of studies we explored patients diagnosed with hereditary neurodegenerative disorders with and without vestibular system involvement. In the first study we explored 18 genetically confirmed Huntington’s disease patients (44.7±8.1 years; male=9). VOR latency, VOR gain and QEM characteristics were not different from controls (p>0.11 for all comparisons), suggesting that VOR is preserved at physiological frequency domains in these patients, even in more advanced stages of the disease. In the final study we explored 23 patients with a clinical and genetically confirmed diagnosis of spinocerebellar ataxia (SCA) type 3 (n=15), type 2 (n=4) and type 1 (n=4]), and 9 patients with early onset Friedreich’s ataxia (FA). VOR latency was higher in FA (p<0.001) and SCA3 (p=0.02) as compared to controls, discriminating FA from other ataxic patients with an overall diagnostic accuracy of 88%. VORr, VOR40 and VOR60 were significantly lower in FA and SCA3 (p<0.01). VOR80 was only significantly lower than controls in SCA3 (p<0.01), discriminating these from other ataxic patients with an overall diagnostic accuracy of 78%. Covert saccades were only triggered in SCA3 but with low occurrence rate and peak velocity (11.1±28.5% and 77.50±15.30°/s) whereas overt saccades were present in all groups. VORr gain showed a negative correlation with disease severity evaluated with SARA (Spearman r=-0.46, p=0.01). vHIT provides phenotypic information that differentiates the most common autosomal ataxias and can serve as a strategy to orient genetic diagnosis. A correlation between VOR and SARA raises the possibility of using VOR gain as a neurophysiologic biomarker for disease severity. Altogether these results supply relevant data in distinguishing peripheral and central nervous system (CNS) vestibular deficits, particularly acute deficits in emergency situation, as acute CNS vertigo can be life-threatening (stroke) and require immediate medical action. We first demonstrated that not only VOR instantaneous gain analysis has topodiagnostic value but also the analysis of gain dynamic changes, as these can point to individual aetiologies, e.g. a SCC plug. Secondly we demonstrated that quick eye movements also supply topodiagnostic cues, and should have their latency, peak velocity, direction and occurrence rate analysed. At a neurophysiological level, the oculomotor responses generated by head impulses also provide an understanding of both the biomechanical cupular-endolymph dynamics, the VOR dynamic control processes taking place and the modulation of vestibular spontaneous nystagmus with head impulsesA vertigem e a tontura são sintomas muito prevalentes que acompanham uma grande variedade de patologias, nomeadamente as vestibulopatias periféricas, centrais e as perturbações somatoformes. Um diagnóstico correcto é o pré-requisito para um tratamento eficaz, o qual deverá ser dirigido à patofisiologia de base. Os métodos neurofisiológicos que testam a integridade dos circuitos do sistema vestibular central e periférico são essenciais para alcançar um diagnóstico preciso na prática clínica. Actualmente, essa avaliação é realizada principalmente pela análise dos movimentos oculares originados pela estimulação dos canais semicirculares, nomeadamente a estimulação calórica e os impulsos cefálicos. A quantificação do parâmetros dinâmicos do reflexo vestíbulo-oculomotor (VOR) bem como a caracterização dos movimentos oculares rápidos (QEM, Quick Eye Movements) desencadeados com os impulsos cefálicos podem agora ser avaliados de forma fácil e nãoinvasiva com o vídeo Head Impulse Test (vHIT). Tal proporciona a oportunidade única de promover estudos neurofisiológicos das respostas oculomotoras desencadeadas pelos impulsos cefálicos em humanos. Acelerações horizontais da cabeça geram, na obscuridade, movimentos oculares conjugados lentos e compensatórios na direção oposta, sendo este reflexo denominado VOR. O principal objetivo deste reflexo é a manutenção de visão nítida e clara por estabilização da imagem na retina, principalmente durante os movimentos rápidos da cabeça. O Head Impulse Test (HIT)1 ou teste de impulsão cefálica é um teste clínico ativo em que este VOR angular é testado a alta frequência. O clínico, ao colocar-se de frente para o doente, aplica movimentos de frequência e direção imprevisíveis segundo o plano horizontal, de baixa amplitude (10-25º), alta aceleração (3.000-6.000º/s2) e velocidade (150-300º/s), enquanto o doente é instruído a manter a fixação num ponto. Se o VOR estiver intacto, o doente será capaz de manter a fixação, não se observando qualquer movimento rápido do olho, denominando-se o HIT de normal ou negativo. Pelo contrário, se o VOR não for compensatório, o olho acompanhará a cabeça durante a rotação impulsiva pelo que no final do impulso será necessário realizar uma sacada de refixação para recolocar o alvo 1 Halmagyi GM, Curthoys IS, Cremer PD, et al. The human horizontal vestibulo-ocular reflex in response to high-acceleration stimulation before and after unilateral vestibular neurectomy. Exp Brain Res. 1990;81(3):479–90. na fóvea, denominando-se o HIT de positivo ou patológico. Dado que não é possível ao olho humano detectar o movimento de fase lenta do VOR durante este impulso, a presença de uma sacada compensatória no final de um HIT clínico é entendida como um sinal indireto de uma fase lenta não compensatória. Enquanto o HIT unicamente permite a identificação da presença de sacadas após o impulso cefálico, o vídeo HIT (vHIT)2 possibilita não só a identificação e a quantificação da fase lenta do VOR, como também das fases rápidas geradas durante e após o impulso cefálico. Indivíduos saudáveis geram fases lentas compensatórias de baixa latência (7-10 ms), gerando fases rápidas ocasionais após os impulsos. Pelo contrário, doentes com lesão vestibular unilateral (UVL) desencadeiam fases lentas com latência aumentada, nãocompensatórias durante impulsos ipsilesionais , assim como movimentos oculares rápidos durante ou após os mesmos. Estes movimentos rápidos são conhecidos como sacadas covert se desencadeadas durante o impulso cefálico, dado a sua observação não ser possível a olho nu, ou como sacadas overt se desencadeadas após o impulso cefálico. Dado que estas fases rápidas apresentam o mesmo sentido da fase lenta deficitária, diminuindo o erro ocular, são consideradas sacadas compensatórias. Os doentes UVL agudos também podem gerar fases lentas não compensatórias durante os impulsos contralesionais, resultado da lesão da via inibitória ipsilesional, bem como gerar fases rápidas. A quantificação do HIT por vídeo-oculografia permite aumentar substancialmente a sensibilidade e a especificidade do HIT na avaliação do VOR sem as dificuldades técnicas dos coils, de difícil utilização na prática clínica. As novas câmaras digitais apresentam características de peso, forma, resolução espacial e de taxa de amostragem que permitem a sua utilização na prática clínica na quantificação do HIT com boas taxas de correlação com o coil . O registo dos perfis de velocidades ocular e cefálica durante o impulso cefálico permite o cálculo do ganho do VOR, definido como o ratio entre estas velocidades. Esse ratio pode ser calculado em momentos específicos, como p.ex. a 40, 60 e 80 ms após inicio do impulso (ganho instantâneo) ou como resultante de regressão linear (ganho por regressão). Enquanto que o último 2 Bartl K, Lehnen N, Kohlbecher S, Schneider E. Head Impulse Testing Using Videooculography. Ann N Y Acad Sci. 2009;1164(1):331–3.parece ser o valor mais robusto, o primeiro permite a avaliação variação dinâmica do ganho do VOR durante o impulso. Para o cálculo do VOR contribui a sua latência, de tal forma que se esta fosse zero deveríamos ter valores de ganho de 1.0 . Dada a existência de uma latência e, portanto, de uma discrepância entre as curvas de velocidades cefálica e ocular, os valores de normalização que obtivemos no nosso laboratório são ligeiramente inferiores (0.95±0.09). Calculando os limites de normalidade do ganho de VOR, obtivemos valores de 0.77 a 1.13. A avaliação do ganho de VOR permite por último o cálculo da assimetria interaural, que apresenta nas nossas séries, valores de normalidade muito baixos (<6.97%), quando comparados com os valores de normalidade para as provas calóricas (<25%). Os QEM são identificados como picos de aceleração bidirecionais e são classificados de acordo com o sentido relativo à fase lenta, a latência (ms), o pico de velocidade (º/s) e a taxa de ocorrência (%, taxa de impulsos que geram esses QEM). Os QEM podem apresentar o sentido da fase lenta do VOR deficitário e contribuir para a diminuição do erro ocular, sendo consideradas sacadas de correção ou sacadas catch-up, em analogia com os QEM da perseguição sacádica. Nas situações em que o ganho do VOR apresenta valores superiores à normalidade (situação observada em doentes com algumas patologias centrais) podem assumir o sentido contrário ao da fase lenta do VOR e ser igualmente classificadas como sacadas de correção uma vez que trazem a retina de regresso ao alvo. Nos indivíduos normais por nós estudados as sacadas overt apresentam valores de velocidade e de taxa de ocorrência relativamente baixos, enquanto as sacadas covert são inexistentes. Assim, a existência de uma lesão vestibular aguda (UVL) é verificável através do vHIT pela presença de uma fase lenta não compensatória durante os impulsos ipsilesionais. O cálculo do ganho do VOR e do índice de assimetria, permitem quantificar o grau da lesão. Nas fase aguda da lesão, o erro ocular resultante de um menor ganho de VOR é mais elevado, pelo que são identificadas sacadas compensatórias mais frequentes, com maior velocidade de pico e maior amplitude, tanto durante como após o impulso ipsilesional. Pelo contrário, durante a recuperação da fase lenta verifica-se o aumento progressivo da latência e diminuição da taxa de ocorrência destas sacadas. A maior parte dos doentes com síndrome vestibular agudo3 , definido como vertigem espontânea com nistagmo espontâneo, náuseas, vómitos e 3 Hotson JR, Baloh RW. Acute vestibular syndrome. N Engl J Med. 1998;339(10):680–5. desequilíbrio, resultam de lesão vestibular unilateral aguda. No entanto, a identificação no serviço de urgência daqueles que resultam de lesões do sistema nervoso central (CNS) e potencialmente em maior risco constitui um desafio. Como a análise isolada do tipo de nistagmo espontâneo não é suficiente para diferenciar os doentes com patologia periférica daqueles com patologia do sistema nervosa central, desenvolveram-se para este efeito algumas provas clínicas. Uma das mais importantes é o HIT. A ausência de sacada de refixação durante os impulsos ipsilesionais em doentes com nistagmo espontâneo e sem evidência de outros sinais e sintomas neurológicos, parece ser o que melhor prevê isoladamente a existência disfunção do CNS como causa do síndrome vestibular agudo4. A presença de nistagmo espontâneo constitui no entanto uma dificuldade adicional dado que as fases rápidas do nistagmo e a sacadas overt apresentam a mesma direção, ambas fazem o reset da fixação ocular e partilham propriedades cinemáticas. A realização de provas adicionais tais como o alinhamento ocular vertical (vertical skew) e sentido do nistagmo na levo e dextroversão aumentam o valor diagnóstico do HIT, mas requerem aptidões e competências habitualmente não disponíveis no serviço de urgência. Dado que o vHIT permite a quantificação das respostas oculomotoras aos estímulos impulsivos e apresenta uma curva de aprendizagem rápida na execução da prova, procurámos realizar um conjunto de experiências com o objetivo de determinar se o envolvimento de vias vestibulares centrais causam alterações do VOR dinâmico objectiváveis com o vHIT. Colocámos como hipótese que tais alterações poderiam ser não só traduzidas num algoritmo para topodiagnóstico clínico mas também contribuir para a compreensão dos mecanismos neurofisiológicos de controlo do VOR impulsivo. Para tal estudámos indivíduos saudáveis, doentes com UVL e nistagmo espontâneo de origem periférica e central e doentes com diagnósticos específicos de doença neurodegenerativa hereditária, com e sem envolvimento das vias vestibulares centrais. Nos próximos parágrafos são sumariamente descritos os fundamentos, objectivos, métodos, resultados e conclusões das experiências realizadas.

Caracterización de tarjeta de audio para adquisición de señales EOG

Los movimientos oculares sacadicos son de gran utilidad en el diaanóstico y evalucaciñon de enfermedades neurológicas, para su adquisición y procesamiento se necesita de equipamiento especializado y caro. en este trabajo se realiza la caracterización de una tarjeta de audio de uso comercial con el objetivo de evaluar si es adecuada como una alternativa de bajo costo para la medición de estos movimientos. Se realizan un grupo de pruebas que incluyen la determinación de la calidad de la forma de onde del generadr de funciones utilizado, así como el rango dinámico de la tarjeta, su lineabilidad, resolución, rspuesta de frecuencias, respuesta a la onda cuadrada y la estabilidad temporal, A pesar de que en algunos de estos parámetros se obtienen resultados muy buenos, las limitaciones presentes en la adquisición de señales de muy baja frecuencia hacen que se concluya que no es factible el uso de este tipo de hardware en la medición de estos movimientos. Keywords:&nbsp; &nbsp;sacadas, &nbsp;movimientos &nbsp;oculares, &nbsp;adquisición de datos, &nbsp;AD

Consensus Paper: Neurophysiological Assessments of Ataxias in Daily Practice

The purpose of this consensus paper is to review electrophysiological abnormalities and to provide a guideline of neurophysiological assessments in cerebellar ataxias. All authors agree that standard electrophysiological methods should be systematically applied in all cases of ataxia to reveal accompanying peripheral neuropathy, the involvement of the dorsal columns, pyramidal tracts and the brainstem. Electroencephalography should also be considered, although findings are frequently non-specific. Electrophysiology helps define the neuronal systems affected by the disease in an individual patient and to understand the phenotypes of the different types of ataxia on a more general level. As yet, there is no established electrophysiological measure which is sensitive and specific of cerebellar dysfunction in ataxias. The authors agree that cerebellar brain inhibition (CBI), which is based on a paired-pulse transcranial magnetic stimulation (TMS) paradigm assessing cerebellar-cortical connectivity, is likely a useful measure of cerebellar function. Although its role in the investigation and diagnoses of different types of ataxias is unclear, it will be of interest to study its utility in this type of conditions. The authors agree that detailed clinical examination reveals core features of ataxia (i.e., dysarthria, truncal, gait and limb ataxia, oculomotor dysfunction) and is sufficient for formulating a differential diagnosis. Clinical assessment of oculomotor function, especially saccades and the vestibulo-ocular reflex (VOR) which are most easily examined both at the bedside and with quantitative testing techniques, is of particular help for differential diagnosis in many cases. Pure clinical measures, however, are not sensitive enough to reveal minute fluctuations or early treatment response as most relevant for pre-clinical stages of disease which might be amenable to study in future intervention trials. The authors agree that quantitative measures of ataxia are desirable as biomarkers. Methods are discussed that allow quantification of ataxia in laboratory as well as in clinical and real-life settings, for instance at the patients' home. Future studies are needed to demonstrate their usefulness as biomarkers in pharmaceutical or rehabilitation trials

The utility of the auditory brainstem response in children with atypical saccadic eye movements

Full version unavailable due to 3rd party copyright restrictionsLesions in the brainstem result in widespread damage to a number of sensorimotor systems including oculomotor and auditory neural circuits. Although these systems are spatially separate and highly specialised, they are also co-located. This thesis, investigates whether lesions in the oculomotor system will also cause co-morbid dysfunction in the auditory pathways. Specifically, we investigated the usefulness of the Auditory Brainstem Response (ABR) in two oculomotor conditions: slow saccades in Gaucher disease (GD) and opsoclonus in Dancing Eye Syndrome (DES). We present four empirical studies. In our first study we systematically investigated the ABR in GD. We found that multimodal testing can better delineate underlying neurological deficits in neuronopathic GD (nGD) and distinguish between phenotypes. In the second study we examined the ABR's utility as a longitudinal, objective marker of disease burden and in a randomised clinical control trial. ABRs continued to deteriorate regardless of treatment. In our third study we assessed audiological function in DES. We found that at least 43% of DES patients have hyperacusis. We also found subtle abnormalities in the auditory brainstem, as shown by the ABR. Our final study explored the onset-offset response in the ABR and assessed its utility as a clinical marker. Overall, this thesis provides new evidence that auditory pathways are also affected in diseases which are traditionally assumed to be ‘oculomotor’ in nature. We believe that there is sufficient evidence to warrant the inclusion of audiological testing, such as the ABR, as part of the standard assessment of newly diagnosed GD patients and that they undergo these tests prior to commencing treatment. These tests may also have a wider application as longitudinal outcome measures for use in clinical trials or as markers of neurological burden in GD and we believe may be useful in other metabolic diseases; we found that current therapies for GD have low efficacy. Understanding the underlying neurological deficits in these debilitating illnesses can only help to improve treatments and the long-term outlook for these patients

A molecular-genetic study of Congenital Nystagmus

Nystagmus is a disorder of eye movement characterised by irregular, uncontrolled and repetitive eye movements. It can occur in a broad spectrum of clinical situations and diseases or it may occur in isolation and an inherited disorder. Surprisingly little is known about the underlying mechanisms of ocular-motor control. Similarly, the pathophysiological mechanisms underpinning nystagmus is also poorly understood. By studying pedigrees in whom nystagmus seems to be inherited as an isolated trait (Congenital Idiopathic Nystagmus), it may be possible to identify some of the genetic causes of this disorder and subsequently understand the pathophysiology.This thesis describes a molecular genetic study of congenital nystagmus. A clinical phenotyping study is followed by linkage analysis and positional cloning. A novel nystagmus gene is investigated in a large cohort of Congenital Idiopathic Nystagmus (CIN) patients and X-inactivation studies are performed. Subsequently, cell culture and RT-PCR work is performed to study expression of this gene. Additionally a pedigree with an atypical congenital nystagmus disorder is investigated and a new mutation within a known cerebellar disease gene is identified.This work contributed to the identification of the first gene for Congenital Idiopathic Nystagmus (CIN). The first detailed temporal expression study of the FRMD7 nystagmus gene was also performed in this study which has directed further studies into the pathogenesis of CIN. Identification of a new mutation in the CACNA1A gene in a pedigree with nystagmus and subtle cerebellar signs has lead to the consideration of this gene in patients who present to hospital with isolated atypical nystagmus

Identification of genetic factors underpinning phenotypic heterogeneity in Huntington's disease and other neurodegenerative disorders

Neurodegenerative diseases including Huntington’s disease (HD), the spinocerebellar ataxias and C9orf72 associated Amyotrophic Lateral Sclerosis / Frontotemporal dementia (ALS/FTD) do not present and progress in the same way in all patients. Instead there is phenotypic variability in age at onset, progression and symptoms. Understanding this variability is not only clinically valuable, but identification of the genetic factors underpinning this variability has the potential to highlight genes and pathways which may be amenable to therapeutic manipulation, hence help find drugs for these devastating and currently incurable diseases. Identification of genetic modifiers of neurodegenerative diseases is the overarching aim of this thesis. To identify genetic variants which modify disease progression it is first necessary to have a detailed characterization of the disease and its trajectory over time. In this thesis clinical data from the TRACK-HD studies, for which I collected data as a clinical fellow, was used to study disease progression over time in HD, and give subjects a progression score for subsequent analysis. In this thesis I show blood transcriptomic signatures of HD status and stage which parallel HD brain and overlap with Alzheimer’s disease brain. Using the Huntington’s disease progression score in a genome wide association study, both a locus on chromosome 5 tagging MSH3, and DNA handling pathways more broadly, are shown to modify HD progression: these results are explored. Transcriptomic signatures associated with HD progression rate are also investigated. In this thesis I show that DNA repair variants also modify age at onset in spinocerebellar ataxias (1, 2, 3, 6, 7 and 17), which are, like HD, caused by triplet repeat expansions, suggesting a common mechanism. Extending this thesis’ examination of the relationship between phenotype and genotype I show that the C9orf72 expansion, normally associated with ALS/FTD, is also the commonest cause of HD phenocopy presentations

Antisense oligonucleotide-mediated therapeutic strategies for neurodegenerative repeat expansion diseases

Over 40 diseases, primarily affecting the nervous system, are caused by expansion of simple repetitive sequences found throughout the human genome, termed repeat expansion diseases. Expansions can occur in coding and non-coding regions of the genome, leading to several proposed mechanisms of disease, accumulation of either toxic RNA or toxic protein, although gain-of-function mechanisms are suggested causes of pathogenesis. Currently, there is no cure nor effective treatment strategy for any repeat expansion diseases. However, for many of these expansion diseases, splice-switching antisense oligonucleotides (AOs) may offer promise as a therapeutic strategy, as these compounds have already demonstrated efficacy in the treatment of other types of genetic disorders. Antisense oligonucleotides are short synthetic nucleic acid analogues, designed to target specific pre-mRNA sequences by reverse-complementary Watson-Crick binding, thereby modifying processing and/or abundance of the transcript and the sequence of the encoded protein. While there are a number of applications for AOs, this study focuses on their utility for preventing translation of toxic protein isoforms, either by altering the target transcript to encode a truncated protein isoform, or by disrupting the reading frame to downregulate endogenous protein production. The first part of this study focused on ameliorating the toxic polyglutamine tract found in the ataxin-3 protein that causes spinocerebellar ataxia type 3 (SCA3). One of nine known polyglutamine disorders, SCA3 is a clinically heterogeneous disease, primarily exemplified by progressive ataxia impairing the speech, balance and gait of affected individuals. SCA3 is caused by expansion of a glutamine-encoding tract located at the 5′ end of the penultimate exon (exon 10) of the ATXN3 gene transcript, resulting in conformational changes in ataxin-3 and a toxic gain-of-function. Here, we describe highly efficient removal of the toxic polyglutamine tract of ataxin-3 in vitro by phosphorodiamidate morpholino oligomers (PMOs). Additionally, these PMOs induced a potentially beneficial downregulation of both the expanded iv and non-expanded protein isoforms. As SCA3 has a typical age of onset in the fourth decade, the observed downregulation could delay age of onset by reducing the amounts of the toxic aggregates. Although we induce downregulation of both isoforms, we believe that the proportion of the truncated protein may be sufficient for overall function of ataxin-3, as some studies have shown ataxin-3 protein to be partially dispensable. Recently, several in vitro and in vivo studies have found that targeted knockdown of transcription elongation factors SUPT4H1, and to a lesser extent SUPT5H, can reduce aggregation of expanded transcripts and protein, and alleviate the disease phenotype in animal models of various expansion diseases. We therefore sought to investigate in vitro, the potential of AO-mediated SUPT4H1 downregulation as a therapeutic strategy. We found that our AOs were able to significantly downregulate SUPT4H1, with minimal changes to the rest of the transcriptome. We then assessed whether this downregulation of SUPT4H1 lead to a reduction in expanded ATXN3 mRNA and/or ATXN3 protein expression, however, unfortunately in the models available and under the current study, no modification to the ATXN3 transcript or protein was observed. This lack of effect may be due to the relatively short, expanded repeat lengths in SCA3 cell lines, and we therefore recommend that future studies assess genes with larger expansions, such as the 100-1000s repeat tracts frequently observed in myotonic dystrophy type 1 (DMPK). In order to create an efficient screening process for finding clinic-ready AOs, it is important to have a detailed understanding of the principles of AO design. We therefore present a comprehensive rationale for efficiently design and in vitro delivery of splice modulating AOs. These approaches and recommendations provide a streamlined methodology for any researcher developing AO therapeutics. The results presented in this thesis indicate that morpholino oligomers will provide superior benefit for the treatment of spinocerebellar ataxia type 3, without the toxic effects that result from other antisense oligomer chemistries. Additionally, AO-induced SUPT4H1 knockdown may yet demonstrate therapeutic v application for a multitude of expansion diseases, pending further investigation into the whole transcriptome effects and in vivo efficacy of this strategy. Lastly, our guidelines for therapeutic AO development should aid other researchers in creating the most efficacious and safe AOs for clinical trials. The work presented in this thesis contributes to the greater body of knowledge about the applications of AOs, as well as the need for reliable and systematic protocols in AO research and interpretation. With ongoing collaboration from our industry partners, Sarepta Therapeutics, there is hope that the work presented here will provide a solid foundation for further research into AO therapeutics for the treatment of neurodegenerative expansion diseases

Network Modeling of Motor Pathways from Neural Recordings

During cued motor tasks, for both speech and limb movement, information propagates from primary sensory areas, to association areas, to primary and supplementary motor and language areas. Through the recent advent of high density recordings at multiple scales, it has become possible to simultaneously observe activity occurring from these disparate regions at varying resolution. Models of brain activity generally used in brain-computer interface (BCI) control do not take into account the global differences in recording site function, or the interactions between them. Through the use of connectivity measures, however, it has been made possible to determine the contribution of individual recording sites to the global activity, as they vary with task progression. This dissertation extends those connectivity models to provide summary information about the importance of individual sites. This is achieved through the application of network measures on the adjacency structure determined by connectivity measures. Similarly, by analyzing the coordinated activity of all of the electrode sites simultaneously during task performance, it is possible to elucidate discrete functional units through clustering analysis of the electrode recordings. In this dissertation, I first describe a BCI system using simple motor movement imagination at single recording sites. I then incorporate connectivity through the use of TV-DBN modeling on higher resolution electrode recordings, specifically electrocorticography (ECoG). I show that PageRank centrality reveals information about task progression and regional specificity which was obscured by direct application of the connectivity measures, due to the combinatorial increase in feature dimensionality. I then show that clustering of ECoG recordings using a method to determine the inherent cluster count algorithmically provides insight into how network involvement in task execution evolves, though in a manner dependent on grid coverage. Finally, I extend clustering analysis to show how individual neurons in motor cortex form distinct functional communities. These communities are shown to be task-specific, suggesting that neurons can form functional units with distinct neural populations across multiple recording sites in a context dependent impermanent manner. This work demonstrates that network measures of connectivity models of neurophysiological recordings are a rich source of information relevant to the field of neuroscience, as well as offering the promise of improved degree-of-freedom and naturalness possible through direct BCI control. These models are shown to be useful at multiple recording scales, from cortical-area level ECoG, to highly localized single unit microelectrode recordings

Genetic Testing for Rare Diseases

Rare diseases, or orphan diseases, are those that individually affect a small number of patients, but taken together affect over 300 million people worldwide. They are characterized by their etiological, diagnostic and evolutionary complexity, important morbi-mortality, with high levels of disability that entail and hinder the development of a normal vital subject, not only in those who suffer from them, but also their families; therefore, a comprehensive social health approach is necessary to address this problem.About 80% of rare diseases have a genetic origin, mainly monogenic; thus, genetic testing is mandatory for the confirmation of clinical diagnostics and to ensure correct genetic counseling. Next-generation sequencing (NGS) has enabled a revolution in genetic diseases, specially in rare diseases. However, their complexity makes diagnoses difficult even with the advent of NGS.In this Special Issue, we present several examples of the complexity of genetic diagnosis for most of these diseases and the consequences that genetic testing implies for genetic counseling. There are examples of the genetic heterogeneity of hearing loss, some metabolic and lisosomal disorders, ataxia, Prader–Willi syndrome, and three comprehensive reviews on syndromic retinal dystrophies, the complexity of the molecular diagnosis of neuromuscular disorders, and the value of genetic counseling before and after a genetic test