As recently as a decade ago, many people believed that the cause and cure for all diseases would be discovered upon the sequencing of the entire human genome. But, like most experiments conducted in the laboratory, this information has led to more questions than answers.
DNA is approximately 99.9% identical from one individual to the next. It is this 0.1% difference that confers a unique phenotype to each individual. In addition to being responsible for phenotypic variation, this "minor" variation among individuals can also promote susceptibility to diseases. Now, many scientists are beginning to associate disease risk with the inheritance of specific variants, or single nucleotide polymorphisms (SNPs).
A SNP is a site where a single base substitution occurs at a frequency of at least 1% in the population. Approximately one out of every 1,900 base pairs in the human genome is a SNP, the most common type of variant identified by the Human Genome Project.
Identifying these variants, or SNPs, is relatively easy when compared with the complexity of determining which variants promote increased susceptibility to disease.
"The impact of the genes is intertwined with impact of the environment; for risk prediction, they cannot be separate islands," said Irene Jones, Ph.D., Lawrence Livermore National Laboratory, Livermore, Calif. "It is highly likely that the functional impact of specific variants will be found to depend critically on the context of other variants in a cell that a person has inherited."
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"There are no wild type people with only normal genes," said Jones. "For example, most people have multiple amino acid substitution variants among the genes in each DNA repair pathway."
Being able to fully repair DNA that is damaged by cancer-causing agents is crucial for cells to be protected against deleterious mutations. To date, about 120 genes whose products are involved in DNA repair specifically have been identified, and a total of 200 to 300 genes are involved when DNA damage recognition, repair, and cell cycle checkpoint genes are included. And it is likely that each DNA repair gene has several variants.
Jones pointed out that in a recent study of 37 DNA repair genes, only four did not have variants; there were an average of four variants per gene with an average frequency of approximately 5% in the U.S. population.
To add to this complexity, Larry Thompson, Ph.D., senior scientist, DNA repair, Lawrence Livermore National Laboratory, raised the possibility that there may be enough redundancy among the repair pathways to prevent the variations from actually affecting the cell repair process.
Genotype-Phenotype Correlation
By identifying people who have such variations in their DNA repair genes, individuals may be warned to make a special effort to avoid various environmental exposures, such as cigarette smoking, because of enhanced cancer risk. Therapies can also be developed to maximize the benefit to the patient; for example, some studies have found that patients with specific SNP variants are more sensitive to treatment with radiation therapy.
But not all scientists agree that identifying "at risk" populations is feasible. "The biology/biochemistry at every level is very complex, and every week new proteins are identified," Thompson said. "Statistically, it looks as if every individual will have numerous variants, and susceptibility to any type of damage will be a sum of the variants. I think the whole concept [of identifying at risk individuals] is very naïve."
Thompson pointed out that two DNA repair genes cloned in his laboratory, XRCC1 and XRCC3, both have common SNP variants that have been reported in the literature to be associated with specific types of cancer. However, when these variants were tested in the laboratory, the DNA repair genes did not appear to have impaired function. "These examples of contradictions raise questions about the significance of the genotype-phenotype correlation approach," said Thompson.
Common Versus Rare Variants
One focus of debate is whether SNP variants that are common in the population are more relevant to cancer susceptibility than variants that are rare. And it is not likely that one person will carry only common variants, said Stephen Chanock, Ph.D., Section of Genomic Variation at the National Cancer Institute. "I believe each individual is likely to fall within the spectrum of rare and common variants," he said.
Some researchers believe that a combination of common SNPs can put an individual at risk for cancer, while others believe that many rare variants are more likely to put an individual at greater risk.
"Intuitively, the more common variants are not going to be the ones with much dysfunction," said Thompson.
By contrast, Jones and Marianne Berwick, Ph.D., Biostatistics and Epidemiology, Memorial Sloan-Kettering Institute, New York, both believe that common SNPs are the most relevant variants. "For epidemiology, the more common SNPs need to be investigated, because it is here that we may see the most public health impact," said Berwick.
Cancer Correlation
Two types of approaches are being used to identify SNPs that are associated with increased cancer risk. The candidate gene approach analyzes individual SNPs that could contribute to cancer susceptibility; historically, this approach has been driven by a biological model, which often identifies a geneenvironment interaction.
New developments in technology have improved the prospects for conducting whole genome scanning; this approach uses regularly spaced genetic markers to identify regions potentially involved in cancer risk by linkage analysis. Follow-up analysis can then look at specific SNPs. "The two approaches are complementary and provide investigators with the opportunity to not only find genetic markers, but also functionally significant SNPs, which can correlate the findings with a plausible, biological hypothesis," said Chanock.
A new database is under development by Chanock and colleagues at the National Cancer Institute that will aid SNP research. The database contains 500 SNPs from about 200 cancer-related genes.
With respect to DNA repair variants, Chanock and colleagues are re-sequencing DNA from 102 individuals and looking for SNPs that have been identified by computer searches or those that have been published in the literature.
Chanock said the database will serve as an excellent resource for studying cancer risk for biologists and epidemiologists alike because each SNP will have been validated and the racial background of each individual will be known.
Future Challenges
Research into SNP variants is still in its early stages, and there are many challenges to be overcome. "There is obviously a need for more information pointing toward altered function of candidate variants before attempting a correlation with cancer," said Thompson. He believes that more informative in vivo assays need to be conducted in which the variant is compared to the "normal" (i.e., most common) protein for biological endpoints such as cell survival, mutation, or chromosomal aberrations.
Jones said she believes that making sense of genetic variant data is comparable to making sense of gene expression data. "Identifying the genotype is not the rate-limiting step. Using it intelligently is," said Jones. "Research must increasingly move toward learning to see and understand genotypes as multidimensional entities, not one dimensional, one gene at a time."
In addition, she believes that a "gold standard" population resource should be a high priority for epidemiologists, and should be used as a national/international resource, similar to other major research tools or resources such as x-ray beams and high-end computers. "Competing hypotheses can only be compared if the same resources are used," said Jones.
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