Genomics and Cancer Prevention
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This case study assumes that the reader has studied the article by Martinez ME, O’Brien TG, Fultz KE et al. Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene. Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7859-64 (Full TextExternal)
Part 1. Sorting out the biology
The article introduces several terms that may be unfamiliar to the reader.
“Single-nucleotide polymorphisms” or SNPs are changes of a single base, a single building block, in the string of bases that make up DNA. DNA, the genetic material, is double-stranded. In this instance, the SNP is change of G to A on the strand of DNA that is designated the “sense” strand.
Question 1: On the partner strand of DNA, the “antisense” strand, what is the corresponding base change?
The G to A SNP in the ornithine decarboxylase (or ODC) gene occurs in an “intron” or a non-coding region of DNA. The DNA in introns does not code for amino acids, so it does not make protein. In this region of the ODC gene, called intron 1, the DNA serves as a binding site for proteins that regulate activity of the gene. Gene activity is in large part determined by how often the gene is transcribed into mRNA: how often the gene is turned on or shut off.
Question 2: The authors present background material describing how intron 1, and specifically the region where the G to A change occurs, is involved in regulation of transcription of the ODC gene. How does this regulation occur?
Question 3: APC is a tumor suppressor gene . What happens when APC is functioning normally?
Question 4: What happens when APC is not functioning normally?
Question 5: What happens with the ODC intron 1 G allele is changed to A?
Part 2. Identifying the research questions
Question 6: What do the authors hypothesize about the ODC intron 1A allele and risk of polyps?
Question 7: How does aspirin fit into the scheme?
Question 8: What do the authors hypothesize about interactions between the ODC polymorphism and aspirin?
Part 3. Evaluating the study design
The authors genotyped 688 of 1304 participants in a randomized trial of adenoma (polyp) recurrence. The original study randomized participants to a wheat bran fiber intervention. However, it is important to note that the analysis in this article is observational: the participants were not randomized to aspirin. Participants in both arms of the fiber trial were combined and information was collected from self-administered questionnaires on aspirin use. It was not stated how many participants failed to provide information on aspirin use. A large percentage (47%) of the participants did not provide a blood sample, and it is not known whether providing a blood sample was related to outcome (polyp recurrence) or aspirin use or ODC genotype.
Genotyping was conducted using a PCT-based method known as 5′-exonuclease allele discrimination or Taqman. Two different DNA probes were made, one for each of the intron 1 alleles (G or A). The probes are labeled with a fluorescent dye that is released during PCR amplification. The validity of the assay is maintained using positive controls (DNA standards of known sequence) and negative controls (buffer without DNA).
Question 9: How was the reliability of the assay determined? Do you think these measures were adequate?
Question 10: How was the association between ODC genotype and polyp recurrence measured?
Question 11: How was interaction between aspirin use and ODC genotype assessed?
Part 4: Assessing the
Table 1 presented odds ratios for aspirin use and polyp recurrence, ignoring ODC genotype. An inverse association was found: adjusted OR = 0.68 (95% CI 0.48-0.98). The association for ODC genotype and polyp recurrence, ignoring aspirin use, was also inverse: adjusted OR = 0.48 (0.24-0.99) for A/A versus G/G genotype.
Question 12: Are the ODC genotypes in Hardy Weinberg equilibrium in the study population?
Question 13: Interpret the p-value for the Hardy Weinberg equilibrium test.
Question 14: How does a Hardy Weinberg equilibrium test serve as a quality control check on genotyping procedures as well as on the underlying study design?
Question 15: Figure 1 presents joint effects where both aspirin use and ODC genotype are taken into account. How would you characterize the joint effects of aspirin use and ODC genotype?
Part 5: Implications of the study findings
Question 16: How do the authors interpret the findings?
Question 17: What are the implications of the findings for public health?
Question 18: Would you recommend that aspirin use for the prevention of polyps only be targeted to persons with ODC A/A genotype?
Question 19: Should persons with the ODC G/G or G/A genotype be targetted for increased screening for polyps and colorectal cancer, for example, through fecal occult blood testing or colonoscopy?
Question 20: What other studies should be done?