Identification of a novel mutation in the CLN6 gene (CLN6) in South Hampshire sheep affected with Neuronal Ceroid Lipofuscinosis

Abstract

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 for NCL 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 however no disease-causing mutation was identified. 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. Bioinformatic tools were initially 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, CLN6 orthologous sequences from other species were initially used for identification of highly conserved regions. Of the five identified CNCS (5’ UTR, 3’UTR and introns 1, 2 and 6) the region upstream of CLN6 and intron 1 were considered priorities for sequencing. Given that the Sanger sequencing method was laborious and time-consuming, and that there was rapid development of technology; the Sanger sequencing approach was abandoned and Next-generation sequencing (NGS) methods utilised for the following studies. The 454 Pyrosequencing NGS technology was used to sequence the complete ovine Bacterial artificial chromosome (BAC) to generate an ovine reference sequence for mutation screening approaches. The first mutation screening approach, sequence capture and targeted sequencing approach failed; however, the second approach involving sequencing of long-range PCR (LR-PCR) products successfully identified the disease-causing mutation. LR-PCR amplification of 14 regions within the ovine genome region spanning the CLN6 and flanking sequences followed by SOLID sequencing-by-ligation NGS method identified the disease-associated 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. 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.