Our collaborators at NC State University, particularly Dr. Phillips, have tackled this project with energy and enthusiasm and made some important advances this year. Specifically, the researchers have:
Gathered tumor samples to go along with DNA samples from some affected Deerhounds. Tumor samples can be used for cytogenetic analysis to identify chromosomal changes associated with osteosarcoma.
Here is a more detailed summary of what’s been done, what’s been learned, and the next steps:
Through a collaborative effort between the Scottish Deerhound Club of America and the North Carolina Animal Cancer Program at NCSU, more than 220 DNA samples from family groups of Deerhounds have been collected and stored in a database since 1998, along with their pedigree information. In addition, samples from osteosarcomas that have developed in some of these hounds also have been frozen for genetic analysis.
This unique resource will be used to study the inheritance model of osteosarcoma as well as to perform initial microsatellite marker screening to identify the chromosomal region associated with osteosarcoma development. These are the first steps to determine the genetic mutation associated with the development of osteosarcoma as well as the means by which to develop a screening test for the presence of this genetic trait in Deerhounds.
Determination of the genetic changes responsible for the high incidence of osteosarcoma in Deerhounds will allow further understanding of the mechanisms underlying cancer in general, as well as identify new therapeutic targets.
As of March 2004, NCSU researchers had collected complete pedigree information for more than 800 Deerhounds, 52 of whom are affected with osteosarcoma. DNA samples have been banked from 229 of these hounds, including 29 with osteosarcoma. Pedigree information and medical histories of each participant has been entered into a commercially available pedigree management program (Cyrillic 2.1.3, Cherwell Software). Complex segregation analysis has been used to estimate the heritability of osteosarcoma within these individuals and determine the most likely model of inheritance. Four models of disease transmission were evaluated, including autosomal dominant, autosomal recessive, polygenic, and environmental. A general class A logistic regressive model as described by Bonney (15) was used to evaluate these four models for significance.
A preliminary pedigree analysis of a subset of Deerhounds strongly suggests that osteosarcoma risk is related to a single genetic locus inherited in a recessive fashion. Assuming an autosomal recessive mode of transmission, NCSU researchers also have performed a power analysis to determine the likelihood of identifying a genetic marker statistically associated with the osteosarcoma phenotype in this group of Deerhounds.
Pedigree structures and disease status were evaluated using the program SIMLINK ver. 4.12. This program simulates expected genetic marker data for a locus linked to the disease causing gene, given the input pedigree structures and disease status. Risk calculations are then performed on the expected marker data and maximum likelihood methods are used to determine their significance. This process is performed in an iterative fashion (10,000 times in our case), the results of each iteration are then used to estimate the likelihood of success (defined in terms of odds ratio).
Using our current sample size and structure, NCSU researchers estimate a better than 10,000 to 1 chance that they’ll succeed in identifying a genetic marker for osteosarcoma in Deerhounds!
The initial genome-wide genetic marker screening will be performed on a subset of the Deerhound population that consists of multi-generation pedigrees with multiple affected individuals. These multi-generation pedigrees are selected because they are considered fully informative, which implies that we can definitively determine the parental origin of a specific genetic marker. Using only fully informative individuals in the initial screening process greatly reduces the number of individuals needed (and therefore the number of experiments and the overall cost) to obtain statistical significance. For example, by using fully informative individuals, statistically significant results can be achieved through studying as few as five hounds for an autosomal recessive disorder and 10 hounds for an autosomal dominant disorder.
Genome screening with polymorphic canine genetic markers (polymorphic markers are pieces of DNA that show some degree of variation within a population and are inherited in a Mendelian fashion) will be performed using the DNA described previously and a PCR-based assay method. The PCR primers (these are the short oligonucleotide sequences from conserved regions flanking the markers that allow for their amplification and detection) for the genome-wide screening will be purchased as a complete set, and have been previously described. This fluorescently labeled screening set consists of pairs of oligonucleotide primers that can be used to amplify >300 polymorphic markers throughout the canine genome. The average spacing of these markers is ~5cM (a genetic measure of distance equivalent to roughly 5 Mb of DNA). Allele frequencies and sizes for these polymorphic markers in the Scottish Deerhound population will be estimated from a group of 20 members of our population (i.e. 40 chromosomes in total). This group of 20 dogs will also be used to identify markers which may be multiplexed (combining several PCR reactions into one) and to optimize their reaction conditions as described previously.
The PCR reaction mixtures (10 ml) will incorporate a fluorescently labeled primer into the product to allow visualization of alleles by automated genoytping equipment (Perkin Elmer Prism 377 96-lane Automated DNA Sequencer), following separation by polyacrylamide gel electrophoresis. Genotype scoring will be performed as described previously. The resultant genotypes will be statistically analyzed using a parametric (where the parameters are Mendelian) method based on the previously determined model of inheritance, to identify a chromosomal region statistically associated with the osteosarcoma phenotype in this subset of the population. The major advantage of using this parametric method in the initial screening process is that it incorporates genotype information from all individuals and thus can generate the most robust estimate of linkage.
Once an area of linkage to the osteosarcoma phenotype is defined in a subset of the population, non-parametric (i.e. allelic association) modeling will be used on the remaining population to further refine the chromosomal region of interest. Allelic association relies on population-level (not pedigree-level) associations between disease loci and nearby marker alleles. A special type of allelic association occurs when a particular marker lies so close to a disease susceptibility locus that the alleles are inherited together over many generations. This type of association is referred to as linkage disequilibrium. Linkage disequilibrium implies that the same allele can be detected in affected individuals in multiple apparently unrelated families.
The number of individuals needed to detect disequilibrium depends on the frequency of the marker allele associated with the disease locus. For this portion of the project the allele frequencies for each of the polymorphic markers that will be defined in a group of 20 dogs becomes important (as described previously). For example, if the marker allele has a frequency of 0.20 in the general population of Deerhounds, an observation that this marker allele has a frequency of 0.80 in affected individuals represents a 400% increase in frequency. Using chi-square analysis, 10 affected individuals would have to be evaluated at this particular allele to show a significant association (p<0.001). The distances being measured by linkage disequilibrium are very small (<1 cM). Thus, this method will allow the fine-mapping of a disease locus previously identified.
To perform this analysis, the same PCR-based assay method will be used as described previously. We will analyze all of the affected dogs and a portion of the unaffected dogs. Genetic markers will be selected from the large region defined in the initial subset of the population. The affected and unaffected dogs will be scored at each of the selected markers to determine their specific genotype. Genotypes will be statistically evaluated using a non-parametric method to detect linkage disequilibrium.
To assist in the process of narrowing the region, we also have access to a canine BAC library. This library may prove useful in the identification of additional markers within the previously defined region of interest. It also may prove useful for the selection of positional candidate genes for further analysis.
Pedigree Analysis: Purchase supplies and materials to perform PCR-based screening of microsatellite markers.
Linkage Analysis: Determine the allele frequencies for a representative set of genome wide polymorphic markers on a subset of the Deerhound population. Verify which markers can be successfully multiplexed and their reaction conditions. The initial PCR-based genome-wide microsatellite marker screening of the selected subset of population will then be performed. Statistical analysis of the results from this screening process and the identification of initial chromosomal region associated with osteosarcoma. Select individuals and markers to be used to further refine chromosomal region through linkage disequilibrium.
Linkage Disequilibrium: Perform PCR-based linkage disequilibrium analysis of the entire DNA sample population. Statistical analysis of these results using non-parametric methods to further refine (narrow) the region of interest. Design a PCR-based test to screen for carriers of the disease gene in Deerhounds—and potentially the general canine population.
Copyright 2004 by Dr. John Dillberger, P.O. Box 910, Creedmoor, NC 27522-0910. Reprinted with permission. All rights reserved.
For more information on osteosarcoma in Scottish Deerhounds, or in other breeds of dog, please click on the links on the right.