The genetic basis for response to the Ketogenic diet in drug- resistant epilepsy Natasha Emma Schoeler A thesis for submission to UCL for the degree of Doctor of Philosophy 1 Declaration I, Natasha Emma Schoeler, 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. Signed: ………………………………………………… Date: 2 Statement of contribution The idea for this thesis was a joint effort between Profs Sanjay Sisodiya, Helen Cross, Ley Sander and I (I am referred to as ‘the researcher’ throughout the thesis). I completed all ethics applications and amendments. All recruitment and collection of blood samples was undertaken by me, with the exception of participants recruited from Bristol Royal Hospital for Sick Children, Alder Hey Children’s Hospital and The Royal Children's Hospital in Melbourne; some participants from Birmingham Children’s Hospital, St George’s Hospital and Matthew’s Friends clinics were recruited by a specialist nurse (Bernie Concannon), a dietitian (Orla Stone) or keto-assistant (Valerie Aldridge) respectively, and some participants were recruited by me. I collected all phenotypic data, with the exception of individuals recruited from The Royal Children's Hospital in Melbourne, for whom Miss Jacinta McMahon provided phenotypic data. DNA collected in the UK was extracted by clinical geneticists at GOSH. I prepared all DNA samples to be sent to various centres for genotyping or sequencing. Miss Anna Tostevin introduced me to the laboratory setting and assisted with some dilutions and packaging. Sequencing of SLC2A1 was completed by Dr Suzanne Drury; sequencing of KCNJ11 and BAD was completed by members of Professor Sian Ellard’s team at Royal Devon & Exeter Hospital; whole exome sequencing was completed by Miss Deborah Hughes and Dr Alan Pittman. Raw genotyping data from the Illumina HumanOmniExpressExome Beadchip was quality- control filtered and exported into PLINK format by Dr Costin Leu. The Reference Sequence Database file provided by PLINK/SEQ, used in exome sequencing analyses, was amended by 3 Dr Costin Leu. The effective number of independent tests and the significance threshold for the genome-wide association study was calculated by Dr Jon White. I performed all other statistical and genetic analyses. 4 Abstract The Ketogenic diet (KD) is an alternative treatment option for people with drug-resistant epilepsy. It can reduce seizure frequency, but it is resource-intensive and may cause adverse side effects. Predictors of response – which, in the absence of specific metabolic disorders, are unknown – would improve patient selection and may enhance understanding of how the KD exerts its antiseizure effect. This project is concerned with identifying possible genetic markers of response to the KD. DNA was extracted from capillary blood taken from individuals who were following the KD for their epilepsy, or who had done so in the past. Individuals were classed as responders if they achieved ≥50 seizure reduction. Response was classified at various follow-up points, as well as a summary of response over time. Association studies were conducted using candidate gene (KCNJ11 and BAD) sequencing, genome-wide single nucleotide polymorphism array, and whole exome sequencing data to determine whether there was an over-representation of specific gene variants in KD responders, compared to non-responders. Common variation in KCNJ11 and BAD was not significantly associated with KD response. rs12204701 reached significance in the array-based genome-wide association study including common variants, with 3-month diet response as the phenotype. No significant results were obtained when summary diet response was taken as the phenotype. Using the gene-based c-alpha test with the exome sequencing data, including all exonic and splicing variants, ANKRD36C reached significance; using a pathway-based count of case-unique alleles test, the ‘ERBB1 Internalisation’ pathway reached significance. No further significant results were obtained from the exome sequencing data when using other gene- and pathway-based tests or when variants were further filtered according to predicted functional consequence. Other genes with large differences in responder/non-responder minor or alternative allele counts are also of interest. It is unknown how these may 5 contribute to variability in KD response. Some common themes were identified amongst the genes and pathways of significance and suggestive significance: cell cycling, apoptosis, glucose homeostasis, neurological processes and triglyceride biosynthesis. It is biologically plausible that these processes influence KD response, although it is likely that many genes play a role. A larger sample size is needed in order to improve power to detect genotypic-phenotypic associations and increase confidence in the importance of the genes of interest. 6 Table of Contents Declaration ....................................................................................................................................... 2 Statement of contribution ................................................................................................................. 3 Abstract ............................................................................................................................................ 5 List of Tables ....................................................................................................................................14 List of Figures ...................................................................................................................................16 List of abbreviations .........................................................................................................................19 Acknowledgements ..........................................................................................................................22 1 Background: Epilepsy, the Ketogenic diet and Genetics ............................................................23 1.1 Epilepsy ............................................................................................................. 23 1.1.1 Definitions ................................................................................................................23 1.1.2 Burden ......................................................................................................................24 1.1.3 Treatment and management .....................................................................................25 1.2 The Ketogenic diet and Epilepsy ......................................................................... 27 1.2.1 The Ketogenic diet – clinical reports of effectiveness .................................................30 1.2.2 Effects of the Ketogenic diet beyond seizure frequency .............................................40 1.2.3 Tolerability ................................................................................................................42 1.2.4 Retention ..................................................................................................................43 1.2.5 Biochemical response to the Ketogenic diet and putative mechanisms of action 46 1.2.6 Are we able to predict response to the Ketogenic diet? .............................................66 1.3 The Ketogenic diet and Genetics ........................................................................ 78 1.3.1 Differential response to the Ketogenic diet ...............................................................79 1.3.2 Differential regulation of gene expression .................................................................83 1.4 Research questions and hypotheses .................................................................. 92 2 Methodology ............................................................................................................................95 2.1 Ethics and recruitment ....................................................................................... 95 7 2.2 Phenotypic data collection ................................................................................. 99 2.3 Genotypic data collection ................................................................................ 101 2.3.1 Research questions 1 and 2 .....................................................................................101 2.3.2 Research question 3 ................................................................................................104 3 Phenotypic results ..................................................................................................................107 3.1 Introduction..................................................................................................... 107 3.2 Methods .......................................................................................................... 109 3.3 Results ............................................................................................................. 112 3.3.1 An overview ............................................................................................................112 3.3.2 Compliance .............................................................................................................117 3.3.3 Ketogenic diet response: seizure frequency at specific follow-up points ..................119 3.3.4 Factors beyond seizure frequency ...........................................................................123