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ORNL technique could rapidly screen for cystic fibrosis gene

OAK RIDGE, Tenn., Nov. 16, 1995 — A new technique that could be used to rapidly screen many people for the defective gene that causes cystic fibrosis (CF) has been developed by the Department of Energy's (DOE) Oak Ridge National Laboratory (ORNL) and the University of Tennessee Medical Center (UTMC).

In a test of samples from 30 persons who have normal or defective forms of the CF gene, the technique was 100 percent accurate, as reported in the journal Rapid Communication in Mass Spectroscopy.

CF is an inherited fatal disease caused by a genetic defect. About four percent of Americans, mostly Caucasians, carry a defective form of the gene, which makes it the most common genetic defect of its severity in the United States. About 40,000 people in the United States have cystic fibrosis.

People with the disease suffer from respiratory and digestive disorders. Because their lungs become covered with a sticky mucus that promotes infection by bacteria, many CF patients require frequent hospitalizations and continuous use of antibiotics and other expensive medications. The total cost of caring for a typical person with cystic fibrosis, who has a median life expectancy of almost 30 years, is estimated at $250,000.

Because each person with CF is the child of parents who both carry defective forms of a particular gene, there is interest in large-scale screening to let people know their chances of having a child with CF.

The rapid screening technique was developed by C. H. (Winston) Chen and Steve Allman, both from the Photophysics Group of ORNL's Health Sciences Research Division, in conjunction with L. Y. Ch'ang, M. Schell, and C. Ringelberg, all of UTMC's Graduate School of Medicine, Department of Medicine; and Dr. Karal J. Matteson, a CF expert at UTMC's Graduate School of Medicine, Department of Medical Biology. They were assisted by K. Tang, a graduate student from Vanderbilt University. Other collaborators at ORNL include Bruce Jacobson, Mayo Uziel, K. L. Lee, M. Doktycz, G. B. Hurst, Scott McLuckey, Michelle Buchanan, and Richard Woychik.

"Our technique uses laser mass spectrometry," Chen said, "and this is the first time that mass spectrometry has been used to diagnose a genetic disease by DNA analysis. One advantage of this technique over conventional analysis by gel electrophoresis is speed-it's at least ten times faster because the whole procedure can be done in minutes, not hours. Another is that it does not use toxic chemical or radioactive materials, which require costly methods of disposal."

In this technique, a laser and a device for separating gene segments by size detect a common defect or mutation in the CF gene-the lack of key genetic material. The absence of three pairs of chemical bases in a specific region of the gene on chromosome 7 is responsible for 70 percent of cystic fibrosis cases.

Chemical bases are the building blocks of DNA, the blueprint for life. The particular sequence and number of these bases, which varies from gene to gene, determines a gene's function in carrying out a life process or transmitting a trait to the organism's offspring.

The causes of cystic fibrosis, long a mystery, are now becoming clear, thanks to advances in biology. Humans have a gene that manufactures a special CFTR protein that helps prevent the buildup of sticky mucus in the lungs. If the gene is defective, it causes cystic fibrosis.

Each gene is made up of two alleles, one from each parent. One correctly encoded allele of the CF gene is adequate for normal CFTR function. But, anyone who has two defective CFTR alleles will have cystic fibrosis. People who have a single defective allele are called carriers; they may pass the defect on to their offspring. Those with two defective alleles have cystic fibrosis.

If two carriers mate and have a child, the probability is 25 percent that the child will have cystic fibrosis. Thus, an accurate, fast screening technique would inform more couples of the likelihood that their future children would be born with CF.

The ORNL-UTMC technique can screen people to determine if they are normal, if they carry a defective CF allele, or if they have CF. For the experiment, the UTMC staff extracted DNA from human hair samples and isolated the part of each CF gene that would contain the known defect if present. They copied this segment millions of times using the polymerase chain reaction (PCR) technique.

The UTMC staff sent to ORNL 30 samples, each a barely visible droplet, for a blind test. The ORNL scientists mixed each DNA segment with a chemical that absorbs laser light. The mixture was vaporized by ultraviolet light from a laser. The electrically charged DNA molecules formed in the vapor were detected in a spectrometer based on differences in size.

Because the segments of defective CF genes have fewer chemical bases, they are smaller and lighter than the segments from normal genes. Since a lighter segment travels faster than a heavier one between the sample plate and detector, the three types of DNA segments can be distinguished by differences in time of travel.

These differences are displayed on a computer screen as spectra-lines with peaks and valleys. This information indicates whether a person has a normal gene, a totally defective gene, or a gene with a defective allele, making that person a carrier. ORNL's identifications of the 30 samples agreed completely with the results of conventional analyses.

ORNL, one of the Department of Energy's multiprogram research laboratories, is managed by Lockheed Martin Energy Systems, which also manages the Oak Ridge K-25 Site and the Oak Ridge Y-12 Plant.