January 26, 2007
Principal Investigator: Patrick J. Venta, Ph.D.
Microbiology & Molecular Genetics
College of Veterinary Medicine
Michigan State University
East Lansing, MI 48824-4320
Introduction
CMO (craniomandibular osteopathy) is an inherited non-cancerous overgrowth of bone primarily occurring in the jaw region of affected dogs, most of which belong to particular terrier breeds. The disease generally disappears about a year after the initial onset at several months of age. It is rarely fatal, but can cause pain to the puppy and a good deal of distress to the owner. We are continuing our efforts to identify this gene, so that breeders can identify carriers by a diagnostic test and then make informed breeding decisions to avoid producing affected offspring. We have excluded many candidate genes in previous work (including one early in 2005, COL1A1, a gene that produces a very similar phenotype in human patients [Gensure et al., 2005]), as well as portions of many chromosomes. Part of this work has been previously published (Housley et al., 2004). The current work is to exclude the remaining regions of the genome using a systematic whole genome linkage approach.
Production of SINE markers
We are using a new kind of genetic marker called SINEs to do the whole genome linkage analysis. We have developed a semi-automated system that allows reasonable throughput and is relatively inexpensive. We anticipate needing to identify several hundred SINE markers that are variable in the three terrier breeds in order to cover all canine chromosomes to complete the scan. So far, 164 SINE primer sets covering the first 18 dog chromosomes have been completely designed and purchased (68% of the genome), and SINEs for the remaining chromosomes have been selected but the some of the design process has not been finished. The design phase should be nearly complete in the near future.
Linkage data on canine chromosomes 1, 2, and 3
We are systematically scanning chromosomes in numerical order (that is, from largest to smallest). In order to minimize the cost of primers, our strategy is to use two successive rounds of primer design – the first to exclude most of a given chromosome, and the second to fill any gaps in coverage caused by uninformative (i.e., low or non-variable) SINE markers. With chromosome 1, all of the chromosome was excluded with the exception of two small gaps that will need to be filled. Most of the markers that we tested for this chromosome turned out to be useful. However, the markers for the first round of chromosomes 2 and 3 were not as informative as those used for chromosome 1 and, although good stretches have been excluded, a much greater amount of these two chromosomes will require the development of additional SINE markers. Although it is possible that these chromosomes might be less variable, overall, for biological reasons it seems more likely that the result of lower marker variability was simply random chance. We plan to fill the gaps on these chromosomes with a second round of SINEs, and if variation is again low (which, again, we view as unlikely), we will use the SNPs that we have identified in other work to cover these chromosomes. In summary, the gene is not on CFA1 (although two small gaps remain to be filled), and it is not on roughly half of CFA2 or CFA3. We expect to exclude a large portion of the genome is the next two months. Patches of most of the other chromosomes were eliminated as containing the CMO gene in past work, and we are now completing the scans for each chromosome with the current work.
Automated SINE assay
We have not been using the automated system up to this point because we wanted to have complete manual control first so that we would be able to verify that the automated system makes accurate calls on a subset of markers. Although we could continue with the manual method, so far our results have shown the automated system produces good genotyping data, and we will continue to verify this for the next few weeks. Our original plan was to use equipment in the M.S.U. Research Technology Support Facility, located a few hundred yards from our lab, to scan our markers in a column-and-row format (in so-called microtiter plates) to test for linkage. However, our department (Microbiology & Molecular Genetics) has recently acquired a very sophisticated instrument (a Typhoon phosphoimager unit manufactured by Amersham Corp.) that is capable of scanning our plates and putting the data into an appropriate electronic spreadsheet format for import into our linkage analysis program. We will use this instrument because it is housed directly across the hall from our main lab, and because the use of the instrument is free to us, thus saving us time and money.
Decision not to request approval for a change in experimental protocol
There have been reports on the development of automated systems to test markers called SNPs (pronounced snips) during the past year. These automated systems have the potential to allow rapid whole genome scanning for disease genes such as CMO. We had considered using this system in place of the SINE marker system that we originally proposed for the linkage analysis work for CMO. However, two factors make it seem more reasonable to continue with the SINE marker system. The first is that the SNP systems (produced by the Affymetrix and potentially by Illumina corporations) require very high quality DNA for accurate genotyping. The samples that we have are buccal (cheek) swabs, and are unlikely to permit direct, accurate genotyping with the SNP systems. We had hoped that a special way (called whole genome amplification [WGA]) could be used to turn our cheek swab DNA into very high quality DNA. Unfortunately, we have been unable to produce any WGA DNA from the CMO swabs using commercially available WGA kits, even after several attempts. The second reason to continue using our SINE system is cost. We calculate that the cost for the SNP system would be somewhat over budget, and although we were prepared to find and use supplementary funding, any failures that might be caused by lower quality DNA might put the project far over budget. For these reasons, we have decided to continue with the SINE markers which, despite the greater amount of effort required, have been consistently reliable for genotyping the CMO buccal swab DNA samples. It is also worth mentioning that although SNP method may very well become the mainstream mapping method for canine genomics, SINEs may be useful markers for narrowing map intervals. This is because SNP system must have a fixed set of markers for each release by the companies (a fairly rare occurrence), whereas SINEs can be easily chosen in a given interval, and easily genotyped using the automated method that we have developed and are using for our linkage scan.
Updated Timeframe
Although we are on track or somewhat ahead of schedule for developing the SINE markers, we are behind in our schedule for testing the markers for linkage to the CMO gene. However, now that our plan is to use primarily the automated SINE genotyping system, we believe we should nearly able to catch up to our schedule for coverage of the whole genome. It is worth noting that the timing of finding the linked marker is dependent upon random chance, given no a priori reason for suspecting any particular region of the genome for containing the CMO gene. Thus, there is a 50/50 chance of finding the gene after screening half of the genome, and in the case of an early discovery of a linked marker it would not be necessary to screen the complete genome. However, we do want to maintain the schedule in case we are extremely unfortunate and do not find the linkage until that the last one percent of the genome is scanned. Given that we may still have gap filling to do by the end of the funded year, it may be necessary to request a six-month extension from the CHF to complete this phase of the work although, again, we may be fortunate and find the linkage still within the planned year.
