Primer-walking sequencing identifies predominantly noncoding TP53 variants in canine cancers

Area of Science:

• Veterinary Oncology
• Molecular Diagnostics
• Canine Genetics

Background:

• Tumor protein p53 (TP53) alterations are common in canine cancers.
• Incomplete characterization is due to sequencing costs and reference genome discrepancies.
• A cost-effective method is needed for TP53 variant screening in dogs.

Purpose of the Study:

• To evaluate a cost-efficient primer-walking approach for TP53 variant screening in dogs with spontaneous tumors.
• To assess the utility of this method for analyzing TP53 regions beyond coding exons.

Main Methods:

• Collected tumor (n=10) and blood (n=16) samples from 26 dogs with suspected neoplasia.
• Used primer-walking PCR and Sanger sequencing to obtain TP53 sequences (exons 2-11, excluding intron 1).
• Compared bidirectional reads to the Ensembl ROS_Cfam_1.0 v114 reference genome.

Main Results:

• TP53 variations were found in 18 of 26 tumor cases.
• Identified one exonic frameshift insertion and 17 noncoding variants (13 intronic, 4 in 5' UTR).
• Recurrent variants in intron 8-10 region were observed in lymphomas and carcinomas across various breeds.

Conclusions:

• Primer-walking Sanger sequencing effectively screens TP53 regions beyond coding exons in canine tumors.
• Matched tumor-normal sequencing is crucial to determine variant origin (somatic vs. germline).
• Further research is needed to evaluate noncoding TP53 variants as potential biomarkers or drivers in canine cancer.