December 14, 2017

A Missense Mutation in Canine CLN6 in Australian Shepherd with Neuronal Ceroid Lipofuscinosis

Neuronal Ceroid Lipofuscinosis (NCL) was studied in the brain of an Australian Shepherd and through the construction of a pedigree of multplie Australian Shepherds.

Neuronal Ceroid Lipofuscinosis(NCL) is a neurodegenerative disorder that occurs in humans and several other mammals, including dogs. “The NCLs are distinguished from other inherited neurodegenerative disorders by the accumulation of autofluorescent lysosomal storage granules in neurons and other cell types, both within and outside the central nervous system.”One of the most notable symptoms of NCL tends to be loss of vision and coordination. An Australian Shepherd developed NCL at 18 months, by 19 months the dog was blind, and by 24 months the dog had to be put down. The disease had rendered it unable to recognize its owner’s voice, eat, and see. It had made it scared of the outside. The dog’s body was donated to the Tennessee Department of Agriculture where the brain was examined. They found enlarged “lateral ventricles and apparent hypoplasia.  Tissue that was formalin fixed was sent to the researchers for analysis."

DNA was analyzed in two methods:

The pieces of tissue were washed in sodium cacodylate, put in Tissue Tek (an embedding compound for cryosectioning) and frozen. The tissues were then sliced and placed on glass slides with cover slips on top. The sections were examined and photographed with a machine that was equipped for epifluorescence illumination. For at least 24 hours, excess sections were incubated with gentle agitation in a mixed aldehyde fixative. Sections were examined with a JEOL 1400 transmission electron microscope.

Twenty sections of paraffin embedded tissue of an NCL affected Australian Shepherd were pooled, deparaffinized and rehydrated. DNA was extracted from these sections. The process was repeated with the tissue of a non-NCL affected Australian Shepherd. Additional DNA samples from non NCL affected dogs were collected from blood samples gathered at University of Missouri Canine DNA Repository. Software was used to show the CLN6 exons. Exons 2, 3, 5, and 7 were amplified using PCR primers and Oligo software. Exons 1 and 4 were amplified with Invitrogen due to their high GC content.

The first method of DNA examination verified that the dog had NCL because it showed a high amount of auto fluorescent material in the brain, predominantly the granular layers of the cerebellum.  All seven CLN6 exons were able to be amplified using the blood from the control dogs, but only five CLN6 exons were able to be amplified (not 1 and 4) using the tissue of the affected Australian Shepherd. 1 and 4 could not be amplified probably due to degradation of DNA.

637 unrelated Australian Shepherds, the 1 NCL-affected Australian Shepherd, and 43 dogs of other breeds were genotyped into homozygotes and heterozygotes using an allelic discrimination assay.


The research showed that the Australian Shepherd studied did indeed have NCL. The brain storage material resembled the brain storage material of those with NCL with the mutation in the CLN6 gene, so the gene was analyzed for mutations. While NCL cause cannot be concretely determined from DNA from a single dog, the analysis of the gene shows a missense mutation, causing the codon for arginine instead of tryptophan. After comparing this missense mutation to the gene of the 637 unaffected dogs, it’s reasonable to hypothesize that this mutation plays a part in causing NCL in Australian Shepherds.

Additionally, comparing the allele’s between the affected Australian Shepherd and a different Australian Shepherd showed that the dogs differ in the allele (one Is homozygous C and the other is homozygous for the wild-type T allele). This says that there might be two different forms of NCL within the Australian Shepherd breed.

Katz, M., Farias, F., Sanders, D., Zeng, R., Khan, S., Johnson, G. and O'Brien, D. (2011). A Missense Mutation in CanineCLN6in an Australian Shepherd with Neuronal Ceroid Lipofuscinosis. Journal of Biomedicine and Biotechnology, 2011, pp.1-6.

Reviewed by: Anneliese Ceisel