Identifying Osteosarcoma Genes in Mans Best FriendArjun Mahadevan, Snaevar Sigurdsson, Elinor Karlsson, & Kerstin Lindblad-TohVertebrate Genomics Biology Group, Broad Institute of MIT and Harvard, Cambridge, MA

Results

Materials and Methods

RottweilerGreyhound

The location of genes. Thick sections indicate exons while thin indicate introns.

Sequencing of Chromosome 11We successfully sequenced Region 2, Region 3, and Region 6.In Region 6, we noticed that three bases were heterozygous, or could be two different letters. Using the reference genome of the dog, we identified where exactly on the genome this heterozygosity occurred.

The heterozygosity of the three bases has two possible haplotypes. Each haplotype codes for a different amino acid, which could potentially alter the protein structure.

Genotyping of Chromosome 11

Background: Region on chromosome 11 has been found associated with osteosarcoma in greyhounds 6 Mb region (from 43 to 49 Mb) including the association peak was sequenced using Illumina sequencing Over 15,000 SNPs and indels (insertions and deletions) were discovered in the region The top associated region includes a known cancer gene CDKN2a/b that is frequently deleted in tumor samples 45 SNPs: newly discovered SNPs from the region were selected for genotyping using Sequenom technology

Experimental Outline:1. Obtain Samples (cases and controls)2. Genotype samples over the 45 SNPs3. Analyze data for trends and correlationsSequencing of Chromosome 11

Background: While sequencing the chromosome 11 region with Illumina HiSeq, certain problematic regions were not sequenced properly during Illumina Sequencing due to the high GC concentration in the DNA sequence or other unknown factors.

We are attempting to sequence six problematic regions. Three regions are displayed below:

Experimental Outline:1. Design PCR (Polymerase Chain Reactions) primers and set up PCR reactions2. Optimize PCR reactions for GC rich or problematic regions 3. Sequence the products using Sanger Sequencing4. Analyze the sequence and find more sequence variants, hopefully those causing osteosarcoma

Obtaining blood samples

Selecting SNPs for Genotyping

Sequenom AnalysisGenotyping of Chromosome 11Sequencing of Chromosome 11Primer 3 screenshotDesign Primers Using Primer3Choosing Chromosomal RegionsSanger SequencingManhattan PlotLocation of SNPs & Genes

Cluster of High Value SNPs Over Predicted GeneGenotyping of Chromosome 11SNP location & P valueCase Control Ratios for Each Block

Haploview SNP CorrelationHaplotype FrequencyArginine: HyrdrophilicAlanine: Hydrophobic

Zoom in on Chromosome 11

Conclusions & Future Directions

Conclusions:

We successfully sequenced three of the six problematic regions using a Platinum Taq & Betaine PCR setup. In Region 6, we found an amino acid changing mutation resulting from heterozygosity found in three sequential bases: the amino acid potentially changes from arginine to alanine. We did not find any variants in Region 2 & Region 3.These results narrow down our region of interest down from the approximately 1.5 Kb-long total amount of DNA which we sequenced, to a specific single amino acid change in a known cancer gene, CDKN2a/b, that is frequently deleted in tumor samples. However, the specific location of the amino acid change is not in a conserved element, compared to twenty nine other mammals.

Future Directions:

1) Attempt to find the same mutation found in Region 6 in other cases and compare DNA from these cases to DNA of controls.2) Further experiment with PCR optimization in order to successfully sequence Regions 1, 4 & 5.Sequencing of Chromosome 11Future Directions:

1) Identify the causal variants and perform functional analysises such as gene expression and epigenetic analysis.2) Follow up in humans with re-sequencing of homolog gene, identification of rare variants and genotyping in case-control cohorts.

Conclusions:

We successfully genotyped 44 out of our 45 SNPs, with an average call rate of 95.7% for all samples. We discovered a haplotype association in the Greyhounds in Blocks 1 and 2, a region that did not show haplotype association in the Rottweiler control dogs. These results narrow our region of interest down from the 120 Kb-long region which we genotyped, to an approximately 60 Kb-long region containing two predicted genes. Interestingly, these predicted genes are very close to the known cancer gene, CDKN2a/b, that is frequently deleted in tumor samples.

Genotyping of Chromosome 11Boxes in red indicate a high LD (linkage disequilibrium) between the two SNPs that meet at the box. We have selected these blocks or identical group of SNPs in both Greyhound and Rottweilers because both blocks show very high levels of correlation. Displayed under the block name are the possible haplotypes possible in each block. The percentage of how common a particular haplotype is given next to the base letters.While the case control ratio for the haplotypes in the Rottweiler were almost identical, in the greyhound, the ratios were more distinct, and they had a low P value.By identifying new risk factors for canine osteosarcoma, our understanding of the corresponding human cancer has the potential to vastly grow. With a better understanding of osteosarcoma, new targets for drug development can be identified that could improve canine and human health.

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