Genetics in Epilepsy Chien-Ning Lo A thesis submitted to the University of College London for the degree of Doctor of Philosophy June 2009 Institute of Neurology University College London 1 I, Chien Ning Lo, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Acknowledgement I would like to take this opportunity to thank all those who have contributed in various ways to this thesis. First I would like to thanks my fabulous supervisor Professor Simon Shorvon, who has been extremely supportive, generous and did every thing possible for me to make me the luckiest student. Without his excellent guidance, warm encouragement, and delightful enthusiasm, this thesis would not have been possible. Prof Nicholas Wood was my secondary supervisor and he was ably assisted in this task by Dr Henry Houlden to whom I owe many thanks for detailed discussions on the genetic aspects of the work and who guided my learning of laboratory techniques. A very special thank you goes to Dr Mary Davis, who led me through the entire journey of genetic linkage analysis, and taught me invaluable lessons as being a scientist; her tremendous contribution cannot be simply put into words. I would like to thank the entire staff of the Neurogenetic Laboratory of the NHNN, especially Dr Vaneesha Gibbons, Dr Andrea Haworth, and Dr Richa Sud who taught a laboratory naïve disaster the basic knowledge and skills to carry out this work; and many thanks to the entire staff who gave me warm encouragements and many lovely moments we shared together. Many thanks to Professor Sheau Yu Teddy Hsu in Stanford University, who helped me with the primer design and PCR and for allowing me to spend time in his laboratory. Another special thank you goes to Dr Caroline Selai, who looked after me and guided me through many difficult times, and has contributed enormously to my personal development as well as giving me career advices. Without her, my years in Queen Square would have been much less fruitful and much less interesting. Thanks to Professor Linda Luxon, Dr Doris Eva Bamiou and Dr Richard Clement, who kindly assisted me and facilitated the saccadic eye movement pilot study. Many thanks to Miss Juliet Solomon, who bravely proposed to read and give me advices on many pieces of this thesis, and many warming encouragements just before my viva. Many thanks to Miss Jane de Tisi, who helped me recruit healthy control subjects. Also many thanks to my friends especially Miss Katherine Woollett, who has always been there for me through difficulties I encountered. 3 A big thank you goes to Miss Eleanor Harris, who helped with many tedious admission procedures and to print the thesis at the last minute. Many thanks to Mrs Muriel Smith, who helped me in finding medical letters and listens to me when I need to talk. Many thanks to all the patients who took part in this study, their courage and contribution cannot be ignored. Many thanks to all the colleagues who shared the office with me during these years, from time to time their great ideas helped me to carry out my work, and their warm support has been an important part of my life. Finally, I would like to say a very very big thank you to my parents and my sister, who encouraged me to go for this PhD and have been an enormous support to me everyday. 4 Abstract This thesis reports a series of investigations to identify the disease loci of three distinct epilepsy syndromes, and also a pilot study to assess the feasibility of using saccadic eye movements as a biomarker of potential interest in genetic studies. The fact that some epilepsy syndromes have a strong genetic tendency has been known for decades, but very few susceptibly genes have been found. I sought to identify disease loci in three distinct epilepsy syndromes: familial temporal lobe epilepsy (2 families), photosensitive epilepsy (1 large family), and Kohlschütter-Tönz syndrome (5 families). Medical histories, seizure patterns and investigations were gathered through interview and case record reviews. The DNA samples were collected and underwent an eight-cM whole genome analysis. Parametric and non-parametric linkage analyses were carried out to these pedigrees. One of the families with mesial temporal lobe epilepsy was considered to have single gene inheritance and three regions of interest were identified. Five genes were sequenced in 5 individuals in this pedigree but no pathological mutations were found. The other family with mesial temporal lobe epilepsy had more complex inheritance pattern and no disease loci were identified. In the photosensitive epilepsy pedigree, our analysis showed polygenic inheritance with one region of interest on chromosome 16, but with no specific genes identified. In the Kohlschütter-Tönz pedigree, a disease locus was identified on chromosome 16 in a region of 30 known genes but no causative gene was identified. These results, both positive and negative are discussed. In the pilot study to assess the feasibility of the methodology of using saccadic eye movement as a biomarker (as biomarker in pharmocodynemic studies), a system for measurement was devised and a control group and patients with widely varied antiepileptic drug measurements were examined (including one patient with phenytoin toxicity). The experiment showed the intra-individual variability of saccadic eye movements was too large to allow small changes to antiepileptic measurements to be easily identified. That seems that the measurement of main sequence will not be useful as a biomarker for pharmacogenomic or pharmacodynamic studies. It is possible that other measurements will be more reliable and these are discussed. 5 Aims This thesis reports studies in two broad areas. The first is a series of investigations in families using advanced linkage analysis; and the second is a pilot investigation assessing the potential of saccadic eye-movements to be a biomarker of the pharmacodynamic actions of antiepileptic drugs, for future use in genetic (pharmacogenomic) studies. 1. Family Linkage Analyses (8 families) In this body of work, I studied 8 families within three epilepsy syndromes (familial temporal lobe epilepsy, photosensitive epilepsy, Kohlschütter-Tönz syndrome). I collected appropriate clinical phenotypic data (from clinical examination, note review and hospital inquiry), carried out the laboratory genetic analysis of the DNA from individuals in this family (preparation, genotyping and sequencing), and devised and conducted the computerised analysis of this DNA using various linkage programmes. The aims of the study were: i. Familial temporal lobe epilepsy (two families). To identify the genetic basis of the familial epilepsy, to determine whether there was a similar genetic cause in the two families, to determine the mode of inheritance in the families, to identify linked regions, to sequence candidate genes within the linked regions and to determine the clinical phenotype associated with any linkage discovered. ii. Photosensitive epilepsy (one large family): To identify the genetic basis of the Photoparoxysmal response (PPR) in a large family with photosensitive epilepsy, to determine the mode of inheritance, and to identify linkage. iii. Kohlschütter-Tönz Syndrome: To identify the genetic basis (linkage) of this rare syndrome (in five families), to determine the mode of inheritance, to identify linkage, to define the clinical phenotype associated with the linked area, and to if possible identify the causative gene. 2. To conduct a pilot study as part of a programme to investigate the potential of saccadic eye movements (main sequence, peak saccadic velocity) to act as a biomarker of value in pharmacogenomic studies of epilepsy. 6 The long-term aim of this work is to determine whether saccadic eye-movements were useful as a measure of the pharmacological effects of antiepileptic drugs – in other words to determine whether they would prove a useful pharmacogenomic biomarker. The hypothesis was that pharmacological effectiveness on epilepsy would be mirrored by pharmacological effects on eye-movements. The specific objective is to determine whether effects on saccadic velocity due to drugs correlated with drug effects on epilepsy in terms of side-effects or efficacy. If the AED effects on saccades did indeed correlate with clinical AED effect, a second phase of the programme was planned to identify whether polymorphic variation in SCN1A (or other candidate genes) could be identified as underpinning the saccadic variation – thus potentially leading eventually to the use of saccadic eye-movements as a biomarker, and identification of genotypes which correlate to clinical response. The first phase of the project reported in this thesis, the pilot (or scoping) phase, which had the following narrower aims: 1. to devise a clinically-applicable method for measuring saccadic main sequence and peak saccadic velocity. 2. to carry out measurements in a suitable control group, to identify mean values and the range of normality. 3. to carry out a pilot study in patients with epilepsy, to determine whether changes in blood levels were reflected by measurable changes in saccadic velocity in a reliable fashion, and to determine the extent of this effect 4. In a preliminary manner, to whether the there is an association between the magnitude of the effect of saccadic variation and clinical effects (side-effects/efficacy). 7 Personal contribution to the work 1. Contribution to design, methodology, laboratory, analysis and writing up a.. The linkage analysis In the genetic analysis project, I was responsible for the overall design of the study and the design of the analytical strategy (in collaboration with my supervisors), the literature review, the recruitment of subjects, the interviews, obtaining consent and collection of blood samples of the two pedigrees with familial mesial temporal lobe epilepsy (B pedigree and the W pedigree).