“Silent” polymorphisms, previously thought to have no functional effect, may play more of a role in drug resistance and response than scientists thought.
Researchers at the National Cancer Institute have recently discovered that SNPs in the multidrug resistance gene, or the MDR1 gene, can change a protein’s ability to bind to certain drugs without altering the protein’s sequence.
“Changes in drug binding may ultimately affect the response to treatment with many types of drugs, including those used in chemotherapy,” the NCI said in a release announcing the study results, which were published in Science Express last month.
SNPs comprise nearly 90 percent of human genetic variations and occur every 100 to 300 bases along the 3-billion-base human genome. SNPs can occur in coding and noncoding regions of the genome. Scientists believe some SNPs can predispose people to certain diseases or impact their response to a drug. Meanwhile, other SNPs — known as synonymous SNPs because they don’t cause a change in the amino acid sequence of the resulting protein — are believed to have no effect on cell function.
These so-called “silent” gene mutations have been long overlooked in the field because researchers thought they had no impact on protein function.
“Silent polymorphisms are extremely common. How many of them in different genes affect function is not known, but our work raises the possibility that some of these, previously disregarded, should be re-evaluated for the possibility that they change the function of the protein encoded by the gene in which they reside,” Michael Gottesman, the study leader and chief of NCI’s Laboratory of Cell Biology, told Pharmacogenomics Reporter this week.
“Since a silent polymorphism doesn’t change the protein coding sequence, it has always been assumed that it would have no effect on protein function,” Gottesman explained. “Our results show that, at least in some cases, it can by changing the rate at which the mRNA is translated and thereby changing the way in which the protein folds.”
According to experts in the field, the results of the NCI study may change prevailing notions about the overall function and impact of SNPs.
In a statement, NCI Director John Niederhuber upheld the study as an “exception to the silent SNP paradigm” that can change scientists’ “thinking about mechanisms of drug resistance” and alter the “whole understanding of SNPs in general, and what role they play in disease.”
Gottesman and his colleagues found that SNPs in the MDR1 gene result in a protein with an “altered ability to interact with certain drugs and pump inhibitor molecules.”
P-glycoprotein, a protein product of the MDR1 gene, expels drug molecules embedded in the cell membrane, thereby contributing to drug resistance in 50 percent of human cancers. P-gp’s pump action prevents the accumulation of such widely use anti-cancer agents as etoposide and Taxol. Additionally, by monitoring P-gp’s function, researchers can also determine how many different drugs are absorbed by or expelled from the cell.
In the Science Express paper, the authors hypothesize that “the presence of a rare codon, marked by the synonymous polymorphism, affects the timing of cotranslational folding and insertion of P-gp into the membrane, thereby altering thestructure of substrate and inhibitor interaction sites.”
To demonstrate the impact of polymorphisms on this pump activity, NCI researchers genetically engineered cells to contain normal MDR1 or a copy of the gene containing one or more SNPs. Through the use of fluorescent dyes, researchers were able to study the pump’s function by observing whether the dye remained in the cell with or without various inhibitors of the P-gp.
“Silent polymorphisms are extremely common. How many of them in different genes affect function is not known, but our work raises the possibility that some of these, previously disregarded, should be re-evaluated for the possibility that they change the function of the protein encoded by the gene in which they reside.”
“This showed that although the SNPs did not change the expected P-gp protein sequence, the presence of one particular SNP, when in combination with one or two other SNPs that frequently occur with it, resulted in less effective pump activity for some drugs than normal P-gp without the SNP,” the study authors said.
Also, the researchers used an antibody to determine how the SNPs affected pump function and found significant differences in antibody binding depending on the presence of SNPs in the protein conformations of the MDR1 genes. This finding indicates “that the shape of a protein is determined by more than its amino acid — or primary — sequence,” the researchers concluded.
“While the same exact protein sequence eventually got made, this slight change might slow the folding rhythm, resulting in an altered protein conformation, which in turn affects function,” Gottesman said.
Silent Mutations’ Role in Targeted Therapy
According to Gottesman, many drugs are unaffected by this functional change in P-gp as a result of the presence of SNPs.
“Screening upfront for such drugs might eliminate some of the inter-individual variation in drug response,” Gottesman said.
Additionally, the correct drug dosage may also be gauged by determining the patient’s haplotype.
“Knowing the haplotype of a patient could help predict what dosage of a drug would be optimal,” Gottesman said. “For some drugs, the pharmacokinetics of the drugs might be altered if the haplotype is present, and this could affect drug dosage given to the patients.”
Gottesman hopes to expand this research to pinpoint the mechanism by which P-gp behaves abnormally in the presence of SNPs.
“We and others will want to know more precisely how this effect occurs,” he said. “For example, why does the protein fold abnormally, and whether similar effects occur in other proteins whose genes carry silent polymorphisms.”