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Identifying disease-causing nonsense mutations throughout the genome
  
By Bijal P. Trivedi

Researchers have devised a method of identifying a group of common gene defects called nonsense mutations. A gene that carries a nonsense mutation produces an abnormally short protein. An estimated one-third of the genetic mutations responsible for human diseases is thought to be due to this type of defect, according to the researchers who published the report.

The approach, called GINI, is novel because the researchers first seek out mutant genes and then determine whether any are responsible for a specific disease. Using GINI, Erick Noensie and Harry Dietz, of Johns Hopkins University School of Medicine in Baltimore, Maryland, were able to detect known nonsense mutations in colon cancer and Sandhoff disease. Their work appears in the current issue of Nature Biotechnology.

To detect nonsense mutations, the body uses a surveillance system, called the nonsense-mediated mRNA decay (NMD) pathway. The NMD pathway detects and destroys pieces of mRNA that have been transcribed from genes with nonsense mutations before the mRNA can be converted into defective proteins that cause disruption in the cell.

Noensie and Dietz used a drug called emetine to disable NMD surveillance, thus selectively preserving the defective mRNA.


When mRNA is produced from healthy copies of the HEXB and MLH1 genes, the mRNA is present in both emetine-treated and untreated cells (A & C). When these genes carry nonsense mutations, the mRNA is only present in the treated cells (B & D).

Using genechips, which can simultaneously measure the activity of thousands of genes, the researchers compared mRNA from emetine-exposed cells with mRNA from untreated cells; mRNA from genes with nonsense mutations should only be found in the emetine-treated cells.

The researchers tested GINI on colon cancer and Sandhoff disease cells, which are known to contain nonsense mutations in the MutL homolog 1 (MLH1) and hexosaminidase B (HEXB) genes, respectively.

Analysis of genechip data revealed that high quantities of mRNA from the MLH1 gene were present only in emetine-treated cells. Though there were 18 other genes that were present in higher quantities than MLH1, the authors emphasize that GINI's strength is to narrow the possible number of genes. When the same experiment was done with Sandhoff disease cells, the HEXB gene ranked within the top 50 mRNAs only present in the treated cells.

"This approach provides a powerful paradigm for DNA diagnostics that focuses on the type of mutation, rather than the type of gene, underlying a disease," writes Michael Culbertson, of the University of Wisconsin, in Madison, in an accompanying article in the same issue of the journal.

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Culbertson, M.R. Sense versus nonsense in DNA diagnostics. Nat Biotechnol 19, 413-414 (May 2001).
 
Noensie, E.N. & Dietz, H.C. A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition. Nat Biotechnol 19, 434-439 (May 2001).
 

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