Thursday, January 04, 2007

 

'Silent mutations' may not always be silent...

A press release from the National Cancer Institute:

A genetic mutation that does not cause a change in the amino acid sequence of the resulting protein can still alter the protein's expected function, according to a new study conducted at the National Cancer Institute (NCI), part of the National Institutes of Health (NIH). The study shows that mutations involving only single chemical bases in a gene known as the multidrug resistance gene (MDR1) that do not affect the protein sequence of the MDR1 gene product can still alter the protein's ability to bind certain drugs. Changes in drug binding may ultimately affect the response to treatment with many types of drugs, including those used in chemotherapy. The results of this study appear online in Science Express on December 21, 2006.

The genetic mutations examined in this research are known as single nucleotide polymorphisms (SNPs) and are very common. Some SNPs do not change the DNA's coding sequence, so these types of so-called 'silent' mutations were not thought to change the function of the resulting proteins.

"This study provides an exception to the silent SNP paradigm by showing that some minor mutations can profoundly affect normal cell activity," said NCI Director John E. Niederhuber, M.D. "These results may not only change our thinking about mechanisms of drug resistance, but may also cause us to reassess our whole understanding of SNPs in general, and what role they play in disease."

Despite success in treating some cancers with chemotherapy, many tumors are naturally resistant to anticancer drugs or become resistant to chemotherapy after many rounds of treatment. Researchers at NCI and elsewhere have discovered one way that cancer cells become resistant to anticancer drugs: they expel drug molecules using pumps embedded in the cellular membrane. One of these pumps, called P-glycoprotein (P-gp), is the protein product of the MDR1 gene and contributes to drug resistance in about 50 percent of human cancers. P-gp prevents the accumulation of powerful anticancer drugs, such as etoposide and Taxol, in tumor cells. The same pump is also involved in determining how many different drugs, including anticancer drugs, are taken up or expelled from the cell.

In this study, researchers led by Michael M. Gottesman, M.D., head of the Laboratory of Cell Biology within NCI's Center for Cancer Research, demonstrated that SNPs in the MDR1 gene result in a pump with an altered ability to interact with certain drugs and pump inhibitor molecules. In order to show that SNPs can actually affect pump activity, the researchers genetically engineered cells in the laboratory to contain either normal MDR1 or a copy of the MDR1 gene that contains one or more SNPs. Then, they used fluorescent dyes to track pump function based on the accumulation of dye in the cell or the export of dye out of the cell with and without various inhibitors of P-gp. 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 P-gp protein sequences and production levels were normal in both the cells that received the normal MDR1 gene and those that received the mutant versions. Therefore, in order to determine how the SNPs affected pump function, Chava Kimchi-Sarfaty, Ph.D., lead author of the study, and co-workers used an antibody that could distinguish between different P-gp structural conformations. They found significant differences in antibody binding consistent with the existence of different protein conformations in the products of MDR1 genes with or without the SNPs. These results indicate that the shape of a protein is determined by more than its amino acid - or primary - sequence.

Like all proteins, P-gp is comprised of amino acid building blocks. While making P-gp, the cell's protein synthesizing machinery knows exactly which amino acids to put together and in which order by reading a copy of the MDR1 gene coding sequence. DNA consists of a sequence of chemical bases, and the code for individual amino acids is represented by specific sets of three adjacent DNA bases called codons. The SNP that Gottesman and his colleagues studied had only one changed base in one codon of the MDR1 gene. Since several different codons can contain the code for the same amino acid, this SNP only altered the gene by converting one common codon to a rare one, but did not change the amino acid for which it coded.

"We think that this SNP affected protein function because it forced the cell to read a different DNA codon than it usually does," said Gottesman. "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."

Since silent SNPs are frequently found in nature, their biological role has largely been overlooked. However, this study raises the possibility that even 'silent' mutations could contribute to the development of cancer and many other diseases.

Original press release "Researchers Find That a 'Silent' Gene Mutation Can Change the Function of an Anticancer Drug Pump" (Posted: 12/21/2006) available via this link

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Related paper from the journal Science:

A "Silent" Polymorphism in the MDR1 Gene Changes Substrate Specificity

Chava Kimchi-Sarfaty, Jung Mi Oh, In-Wha Kim, Zuben E. Sauna, Anna Maria Calcagno, Suresh V. Ambudkar, Michael M. Gottesman

Abstract

Synonymous Single Nucleotide Polymorphisms (SNPs) do not change the coding sequences, and, therefore, are not expected to change the function of the protein in which they occur. Here, we report that a synonymous SNP in the Multidrug Resistance 1 (MDR1) gene, part of a haplotype previously linked to altered function of the MDR1 gene product, P-glycoprotein (P-gp), nonetheless results in P-gp with altered drug and inhibitor interactions. Similar mRNA levels and protein, but altered conformations were found for wild-type and polymorphic P-gp. We hypothesize that the presence of a rare codon, marked by the synonymous polymorphism, affects the timing of co-translational folding and insertion of P-gp into the membrane, thereby altering the structure of substrate and inhibitor interaction sites.

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General info from Mutations: The start of the evolutionary process:

"Silent mutations are a type of substitution mutations in which a base substitution in the DNA template results no change in the amino acid. This is because the substitution simply resluts in another codon for the same amino acid. Thus these mutations are silent, since you cannot detect them by looking at the protein's sequence of amino acids."

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