December 11, 2008

Update on CA research in Kelpies at the University of New South Wales. 

(Dec 2008)

Our initial attempt to use new technology (the SNP arrays funded by the donation from Terry Snow) to find the ataxia gene in kelpies encountered several problems. We found we had to reassess which of the dogs that we had tested were affected. Then an initial result identifying the location of the gene was thrown into doubt by experiments meant to confirm it. This required an unexpectedly large amount of time and computing to resolve. It lead to the confirmation of the initial predicted ataxia gene location after backtracking from what we thought was a false trail, only to have to retrace our steps as the direction was right after all. This unfortunately caused unexpected delays.

The ataxia mutation has now been localised to a region of 5 million bases (0.2% of the dog genome). There are 44 genes in this region, and at first glance, none stand out as likely to be involved in ataxia. Two candidate genes have been sequenced by checking each of their 10,000 protein coding bases, but no disease causing mutations were identified. We have identified a very unusual gene that is a processed (shortened) copy of a gene found elsewhere in the dog genome which is known to be associated with ataxia in human and mouse. The full original gene product binds the RNA of an ataxia gene in humans called spinocerebellar ataxia-1 (SCA1). The question is does this processed gene copy do anything in the dog?  Is it active?  Too much of the original gene product does cause ataxia in mice. It is possible that this gene is turned on in kelpies with ataxia when it should not be. The 2,500 bases of DNA sequence of the processed gene is not different between affected kelpies and controls, but is there a difference in the control region the front of the gene that has signals to turn it off and on?  If this inactive copy of the gene has become activated, for example by the insertion of a jumping gene just before the start, it could result in ataxia. Two approaches are being taken to investigate this.  One is to examine the activity of this gene by seeing if it makes RNA, the other is to look at the DNA sequence surrounding the gene and see if there are differences between affected's and controls. We are looking at the 19,000 bases before the gene at the moment.

Our Department has recently ordered a $500,000 high throughput DNA sequencer which could allow for each base in the 5 megabase region to be checked in a single experiment. It requires a special capture array to separate the DNA that is from our target region from the rest of the dog DNA, and we are looking at designing this. We will use this approach if we cannot find evidence that the processed gene copy is the cause of ataxia in the Kelpies. The region is complex with many repeat DNA sequences which will make it challenging. In the end we need to find a difference in the DNA of ataxia affected dogs that is not in unaffected controls.

Jeremy Shearman

Alan Wilton