COLUMBUS, Ohio - Researchers have developed a new technique that promises to significantly improve the accuracy of genetic testing for cancer and many other diseases. The process, published in the journal Nature, could significantly improve the diagnosis of mutations responsible for most hereditary cancers, such as retinoblastoma, hereditary breast cancer, and hereditary colon cancer.
It could also be important for helping to determine whether a mother carries a gene mutation on one of her X-chromosomes that could cause a disease on one of her sons. Such so-called "X-linked" disorders include hemophilia and Duchenne's muscular dystrophy.
"Detecting X-linked mutations in females is a diagnostic nightmare that people who do genetic testing wrestle with almost daily," said Albert de la Chapelle, director of the Human Cancer Genetics Program at Ohio State University's Comprehensive Cancer Center.
The technique involves separating pairs of human chromosomes and dispersing them into different mouse cells.
For the present study, the researchers applied the new technique to 10 individuals suspected of having hereditary nonpolyposis colon cancer (HNPCC), one of the most frequent forms of hereditary colon cancer.
All 10 people had a family history of HNPCC and showed other criteria supporting a HNPCC diagnosis, but conventional genetic testing revealed no HNPCC-linked mutation in any of them.
"When we applied this new method, it was an eye-opener," said de la Chapelle. "Every one of the ten individuals we tested was shown to have a key mutation. I would have predicted that maybe three, four, or even five would have been explained, but all ten had a mutation."
The finding means, said de la Chapelle, "that there are a lot of mutations that we are missing." The finding was particularly satisfying for another reason: Of the ten people included in the study, one was a member of the family that triggered the first research into hereditary colorectal cancer in 1913, and in whom HNPCC was first described. But the mutation in this family had previously eluded detection.
"Dr. Henry T. Lynch is the person who has mainly studied this family clinically," said de la Chapelle of the clinician-researcher who first described HNPCC. The condition is also known as Lynch Syndrome. "He, Dr. Bert Vogelstein, and we have been laboring for many years to find that mutation. We knew it was there, but we couldn't find it - until now."
Both Lynch and Vogelstein are co-authors on the paper, along with de la Chapelle and other researchers. The technique is not a substitute for existing genetic tests; rather, it can greatly increase the sensitivity of these tests. That is, it promises to reduce the number of false negative results produced by these tests, particularly for so-called dominantly inherited disorders.
The problem that genetic tests sometimes cannot overcome arises because most cells in the body contain two complete sets of chromosomes. One set comes from the mother, and one set comes from the father. Thus, there is a chromosome 3, for example, from the father and another from the mother. Furthermore, every gene on the paternal chromosome 3 has a counterpart on the maternal chromosome 3.
In some genetic diseases, both copies of the gene must be damaged - mutated - for the disease to occur in offspring. These are known as "recessively inherited disorders." The mutations responsible for these disorders are readily detectable by conventional testing because both copies of the gene are mutated.
But that's not the case with so-called "dominantly inherited disorders." In these diseases, the disorder can occur when only one of the two genes is mutated. This presents a problem for many genetic tests because the presence of the normal gene obscures the presence of the mutated gene.
The new technique separates the two copies enabling each to be analyzed independently. It does this by fusing human cells with mouse cells such that in some fusions only one human chromosome of a pair enters the same mouse cell. In this way, the human chromosomes can be analyzed for mutations independently, thereby preventing a normal gene from masking the presence of a mutated partner.
The primary drawback to the method is the complexity of the process. The cell fusion step is followed by cloning of selected cells and culturing them until enough cells are available for testing. The entire process takes two to four weeks. It also requires fresh, human cells (no older than 48 hours). With time, it could become streamlined and made less expensive, however. Presently, the method is a valuable research tool. For example, human genes that express messenger RNA and produce protein before the fusion process continue to do so after they enter the mouse cell.
"This gives us a wonderful tool for all sorts of experiments," said de la Chapelle. "We can study the activity of a single human gene without the influence of its partner gene."
Contact: Albert de la Chapelle, (614) 688-4781; delachapelle-1@medctr.osu.edu
Written by Darrell E. Ward, (614) 292-8456; Ward.25@osu.edu
Journal
Nature