Quick Update from Dr Neff

[Laurae]

Thanks for the update, Mark. Does this mean dogs previously identified as having the genetic marker are no longer positively identified as such?

[Liz P]

I sure hope it doesn't take 20 years. Our technology is improving exponentially every day...

[Pam Wolf]

In the meantime why not be open about the dogs that are producing EOD? Why do some breeders 'hide' the EOD with pathetic escuses-I shot birds over his head or similar? It is NO ONE'S fault the dog is deaf, it IS a problem we as breeders need to be upfront about so people can make informed breeding decisions.

[Mark Billadeau]

Thanks for the update, Mark. Does this mean dogs previously identified as having the genetic marker are no longer positively identified as such?

There is a recessive gene which is responsible for EOD.

The general location of this gene within the 2,500,000,000 base pair canine genome has been narrowed down to a 3,000,000 locus in the genome.

This evidence was clearly demonstrated by an assocaition study of EOD and normal dogs (see below).

 

Typically, the next step followed by Dr Neff's lab would have located a recessive gene to a very narrow section of the 3,000,000 base pair locus; this has not happened.

 

 

 

This is from the SNP link I posted above.

Each person's genetic material contains a unique SNP pattern that is made up of many different genetic variations. Researchers have found that most SNPs are not responsible for a disease state. Instead, they serve as biological markers for pinpointing a disease on the human genome map, because they are usually located near a gene found to be associated with a certain disease. Occasionally, a SNP may actually cause a disease and, therefore, can be used to search for and isolate the disease-causing gene.

 

To create a genetic test that will screen for a disease in which the disease-causing gene has already been identified, scientists collect blood samples from a group of individuals affected by the disease and analyze their DNA for SNP patterns. Next, researchers compare these patterns to patterns obtained by analyzing the DNA from a group of individuals unaffected by the disease. This type of comparison, called an "association study", can detect differences between the SNP patterns of the two groups, thereby indicating which pattern is most likely associated with the disease-causing gene. Eventually, SNP profiles that are characteristic of a variety of diseases will be established. Then, it will only be a matter of time before physicians can screen individuals for susceptibility to a disease just by analyzing their DNA samples for specific SNP patterns.

 

Dr Neff's research has completed the association study. They are working on finding the exact location of the gene and then the sequence of this gene which would allow them to develop a DNA test analogous to the CEA test.

[Pearse]

There is a recessive gene which is responsible for EOD.

The general location of this gene within the 2,500,000,000 base pair canine genome has been narrowed down to a 3,000,000 locus in the genome.

This evidence was clearly demonstrated by an assocaition study of EOD and normal dogs (see below).

 

 

The "gene" can cover the entire 3 million base pairs of DNA, or some subset of that with the rest being control regions that turn the gene on and off at the proper time in the proper cells, or there could be several genes in that region.

 

Genes in most eukaryotes (organisms whose cells have nuclei, which includes most things not bacteria) have genes made up of regions of DNA that code for part of the protein (exons) and parts of DNA between the eons that do not code for protein (introns). Additional regions encode control regions to turn the gene on and off.

 

Mutations can be of many types.

 

The mutation in CEA is a largish deletion. A big chunk of DNA is missing. That's easy to find, and easy to test for. In the case of CEA, you do a PCR reaction where the primers are either side of the deletion. The product you get in affected dogs is smaller than the one you get in normal dogs. This shows up easily on a gel (there are other more sophisticated ways of doing this now but the principle is the same)

 

Point mutations mean that one DNA base is changed. That can have no effect at all since there is redundancy in the genetic code ( different combinations of bases can code for the same protein building block, or amino acid), can change the protein sequence by one amine acid (this is what happens with scrapie resistance in sheep and the "QR" phenotypes), can truncate the protein, or can change the splicing between introns and eons. (introns are spliced removed, exons spliced together - the way in which this happens can make different products from the same "gene" or coding region"

 

Between any two who are not twins, you are going to see multiple differences in 3 million bases of DNA. Most will be of no consequence (useful in tracking parentage perhaps - DNA 'fingerprinting - but no functional changes). It seems like the Neff group has identified 100 or so of these between the affected dog and the two normal dogs they have sequenced (keep in mind that even five or six years ago the thought of sequencing three whole dog genomes to look for a mutation in one gene would have been preposterous - that is how fast the technology is progressing - the first human genome was sequenced between 1995 and 2003 and cost $3 Billion to do. The cost is now approaching $1000/genome). Now they will need to determine which of those 100 mutations are important in causing the problem, and which are not. Computer analysis will help with some of that by eliminating "silent" mutations that cause no change in the coding sequences.

 

So, trickier to find and trickier to test for. If the mutation just turns the gene off, then no problem. Easy to design a test. If it terminates the protein coding region early, not hard either. If it changes just one base and therefore, changes one amino acid in the protein, trickier. However, that is the case with scrapie resistance in sheep and there is an easy PCR test for that, so it's doable.

 

It should not take 2 years to resolve but that depends on what resources they can devote to the problem full time, and that depends on lab priorities and funding. This lab is not primarily concerned with canine health. It is a human cancer and neurological disease lab who are doing the dog work to build models for human disease (dogs are good because of breed differences, very good pedigree information, multiple offspring, and shorter generation times). We, the dog community, are not funding this research (other than the parts being funding by NIH which is tax-payer funded).

 

In addition, the lab just moved a few years ago, so it has taken Dr. Neff time to set up his facilities, his team, and the infrastructure (computing systems etc) to manage all of his projects.

 

My guess is we will see fairly rapid progress from here. Although three million base pairs sounds like a big region, it really isn't. The canine genome is around 3 Billion base pairs so this is 0.1% and they already have it sequenced three times.

 

Thanks to Mark for taking the time to follow-up with Dr. Neff, and to the Neff group for forging ahead. Keep your fingers crossed.

 

Pearse