COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Copyright Service. sydney.edu.au/copyright IDENTIFICATION OF A NOVEL MUTATION IN THE CLN6 GENE (CLN6) IN SOUTH HAMPSHIRE SHEEP AFFECTED WITH NEURONAL CEROID LIPOFUSCINOSIS Izmira Farhana Mohd Ismail A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Faculty of Veterinary Science University of Sydney Australia February, 2015 SUMMARY Neuronal ceroid lipofuscinoses (NCL/Batten disease) are a group of fatal inherited neurodegenerative diseases that occur in many species including humans, sheep, dogs and cattle. Typical NCL symptoms include progressive loss of vision, regression of mental and motor development, epileptic seizures and premature death. Currently there is no effective treatment or cure for NCL, with the underlying disease mechanisms still poorly understood. Advances in molecular genetics in recent years have allowed the characterisation of hundreds of causative mutations and polymorphisms in at least 17 disease-causing genes across all species. For some species, research colonies have been established for studies relevant to the corresponding human NCL variants. Best characterised of all animal models is the New Zealand South Hampshire (SH) sheep which is a model for the human variant late-infantile form of NCL (vLINCL). Past studies have revealed the ovine CLN6 gene (CLN6) as a strong candidate gene for this disease in South Hampshire sheep. No disease-causing mutation was identified in the gene coding region, however quantitative PCR revealed reduced CLN6 messenger RNA (mRNA) expression in affected sheep compared to normal healthy sheep. The main objective of the present thesis is the identification and characterisation of the mutation responsible for NCL in the South Hampshire sheep. It was proposed that the mutation lies in the non-coding regions within or flanking the gene and that this mutation affects gene regulation. In Chapter 3, bioinformatic tools were used to identify conserved non-coding sequences (CNCS) which are deemed potential regions of interest for regulatory mutations. Due to the limited ovine genome resource available when the study was commenced in 2006, orthologous sequences from nine other species (mouse, rat, human, cattle, macaque, dog, opossum, chicken and fugu fish) were aligned against sheep using the VISTA suite of programs to detect CNCS. These analyses resulted in the identification of five highly conserved regions in the 5’ UTR, 3’UTR and introns 1, 2 and 6 of the CLN6 orthologs. Of the five identified CNCS, the region upstream of CLN6 and intron 1 were considered priorities for sequencing as they were more likely to contain transcriptional regulatory elements and had not been sequenced previously (region upstream of CLN6) or only partially sequenced I (intron 1) in sheep. An ovine BAC clone containing these CNCS regions was used as template for sequencing using the conventional Sanger method and generated 1,450 bp new ovine sequence (Chapter 4). Given that the Sanger sequencing method was laborious and time-consuming, and that there was rapid development of the next-generation sequencing (NGS) technology; the Sanger sequencing approach was abandoned and NGS utilised for the following studies. In Chapter 5, 454 Pyrosequencing NGS technology (Roche) was used to sequence the complete ovine BAC to generate a reference sequence for mutation screening approaches. NGS sequencing of the ovine BAC method was successful and generated approximately 120kb of normal sheep genomic sequence at ~14X coverage that spanned CLN6 and flanking genes CALML4, FEM1b and PIAS1. Two distinct mutation screening approaches were implemented using consensus sequence obtained from alignment of the ovine 454 NGS sequence (Chapter 5) with known sheep sequences from published and unpublished sources. The first mutation screening approach, sequence capture and targeted sequencing approach (Chapter 6) failed; however, the second approach involving sequencing of long range PCR (LR-PCR) products (Chapter 7), successfully identified the disease-causing mutation. The sequence capture approach isolated and enriched a specific ovine genomic region using hybridization on the customised probes on the μParaflo microarray chip (LC Sciences). This method appeared to have not captured the targeted sequence successfully, with only 10% of captured sequences mapping back to the reference sequence. LR-PCR amplification of 14 regions within the ovine genome region followed by SOLID sequencing-by-ligation NGS method (Applied Biosystems) identified the disease-causing mutation as a 402bp deletion and 1bp insertion in ovine CLN6, namely g.-251_+150del and g.+150_151insC. The mutation is predicted to lead to the deletion of the whole of exon 1 and the ATG start codon as well as flanking non-coding sequence. Identifying the disease-causing mutation for NCL in SH sheep provides the long-awaited confirmatory evidence that ovine CLN6 is the causative gene for NCL in SH sheep. It was anticipated that discovery of the mutation would lead to development of a DNA test to screen the SH sheep flock and a wider population of sheep. However, developing a direct DNA test using PCR has been difficult. This is because the identified deletion is in a region that is extremely GC rich and composed of highly repetitive DNA, thus the PCR-based test II produced inconsistencies in amplification and preferential amplification of one allele relative to the other in carriers. However, there might not be a great need to generate a commercial DNA test for the SH sheep as the breed is unique, not widely used for production and localised only in New Zealand. Nonetheless there is a continued effort towards developing a direct DNA test to enable more reliable and effective screening of this mutation in the current SH experimental flock. The information gained in this research confirmed that South Hampshire sheep represent a model for the CLN6 variant in humans. Future research in this large animal model will allow for more effective strategies for developing therapeutic approaches for NCL in humans and further strengthens the invaluable role of this animal model for NCL studies. III DECLARATION I certify that the content of this thesis is the result of work I have conducted since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I certify that to the best of my knowledge any help received in preparing this thesis, and all the sources used, have been properly acknowledged in this thesis. ---------------------------------------------------- Izmira Farhana Mohd Ismail IV ACKNOWLEDGEMENTS First and foremost, I gratefully acknowledge the scholarship bodies: Universiti Putra Malaysia (UPM), Malaysia Ministry of Higher Education and the Faculty of Veterinary Medicine, UPM for giving me the opportunity and financial support to pursue a PhD in Australia. I would like to convey my sincere gratitude to my supervisor Assoc. Prof. Imke Tammen for her outstanding supervision and contributions of ideas, time and funding towards my PhD journey. I am especially grateful that over the years she has not only been a supervisor to me, but also mentor, colleague and friend. I find her diligence and dedication to the genetics discipline and teaching truly inspirational and I aspire to be an academician and researcher like her. A heartfelt thank you to my associate supervisor, Prof. David Palmer from Lincoln University, Christchurch, New Zealand, for his guidance, review of my thesis and for providing valuable insights from his decades of involvement in NCL research. I truly appreciate that Dave and his wife Jeanette offered their home and warm hospitality whenever there was a need to conduct research at Lincoln University. I thank Dr. Michelle Hyde for editorial proof reading of Chapters 2 to 8 and Prof. Frank Nicholas for his continuous support in the genetics field. I would also like to thank Nadia Mitchell at the Lincoln University, Christchurch who has been very helpful, particularly in providing DNA samples and related information from the South Hampshire sheep flock. Her collaboration has made it possible for my research to progress. I would also like to thank my colleague and friend Peter Houweling for taking me under his wing early in my candidature