Rapid Sequence Gene Test Gives Accurate Diagnosis of MD

University of Utah researchers have developed a test that detects the most common form of muscular dystrophy in at least 95 percent of cases-a far higher success rate than the currently available test-and at a feasible cost.

The new test, Single Condition Amplification/Internal Primer sequencing (SCAIP), detects Duchenne muscular dystrophy (DMD) in the 35 percent of cases that current testing misses. Accurate diagnosis spares children with DMD unnecessary invasive procedures and gives parents and patients proper and timely information about the disease, according to U of U neurology and genetics researchers.

SCAIP, which will be available at the University of Utah Muscular Dystrophy Clinic by April 1, allows physicians and geneticists to perform direct sequence analysis of the entire gene to find small genetic mutations that confirm DMD. Until now, many researchers thought using direct sequencing would be too expensive to test for DMD or other diseases.

It was the University of Utah's role as a primary investigation site for the Human Genome Project that gave researchers here the experience to devise an affordable way to use direct sequencing to test for DMD.

"We've developed the ability to rapidly look for genetic variations in the entire gene," said Kevin M. Flanigan, M.D., assistant professor of neurology at the U School of Medicine. "In combination with previous tests that show large duplications in the gene, it will allow us to detect essentially all mutations in the gene."

Along with DMD, the new test detects mutations responsible for Becker muscular dystrophy, a milder and less common form of MD. It also allows clinicians to provide improved genetic counseling, because this testing method detects mutations in the many female carriers whose mutations were undetected by other commonly available techniques.

The U researchers will publish their findings online in The American Journal of Human Genetics. The hard copy of the study will appear in the journal's April edition.

Muscular dystrophy occurs when a mutation in the dystrophin gene on the X chromosome interferes with the production of the dystrophin protein. Duchenne muscular dystrophy happens once in every 3,500 live births and occurs only in boys. The disease, which weakens skeletal and involuntary muscles, appears as early as age 3 when children often exhibit problems walking. By age 12 boys with DMD can no longer walk and typically die by their early 20s because of respiratory and cardiac dysfunction.

Women are carriers, but do not get DMD.

The current commonly available test confirms DMD by looking for missing exons on the dystrophin gene. An exon is the region within a gene that contains the genetic code. In around 60 percent of DMD cases the gene mutation is caused by the deletion of one or more exons.

The current method finds DMD by examining the dystrophin gene within "hotspots," so that checking 2 exons results in the detection of around 98 percent of DMD cases caused by exon deletion. Other tests are available that detect exon duplications, responsible for another 5 percent of DMD cases.

But DMD also occurs when exons are not deleted or duplicated, and because of this the currently available tests fail to detect the gene mutation in 35 percent of cases, according to Flanigan.

"That leaves a large percentage of boys who need a biopsy to confirm the disease," he said.

The dystrophin gene is the largest one in the body, containing 79 exons spread over 2.2 million base pairs of DNA. The gene's size has made it uneconomical to test for genetic variations on a large scale.

To find the genetic mutations, Flanigan and other U researchers use a polymerase chain reaction (PCR), followed by direct sequence analysis. PCR is used to make many copies of DNA fragments, and once enough fragments are made, researchers look for sequence mutations. But using PCR followed by direct sequence analysis of the entire gene has been considered impractical and too costly to test for DMD because it involves analyzing each exon under separate conditions.

The U researchers solved that problem by developing a way to test all exons under the same conditions. The use of a single set of conditions allowed them to automate and miniaturize the PCR and sequencing steps, making the test economical.

The U's work on the Human Genome Project gave researchers a big advantage in developing SCAIP, according to Robert W. Weiss, Ph.D., associate professor of human genetics and head of the Utah Gene Depot. Weiss worked on the Human Genome Project and the U researchers developed SCAIP through his lab.

"The mutation tests that a lot of people thought were going to be difficult, it turns out, aren't that challenging," Weiss said. "It is our practical experience in large-scale sequencing that allows us to do this."

The Utah Gene Depot will conduct the DMD testing for the U's Muscular Dystrophy Clinic. The test will be available for less than $1,000 to examine more than 110,000 base pairs of DNA, according to Flanigan.

In addition to several types of MD, the test will have applications for other diseases, according to the U researchers.Development of a reliably accurate DMD test was long overdue, said Pat Furlong, executive director of the Parent Project Muscular Dystrophy.

"This is monumental work," Furlong said, "and we are grateful for their research."

Furlong's sons died of DMD at ages 15 and 17, and even though her husband is a physician, diagnosing their boys' disease was an ordeal. She hopes the new test will spare other parents that pain, and because the test can identify DMD carriers, people will be able to make better-informed decisions about having children, Furlong said.

The director of research development for the Muscular Dystrophy Association commended the test as an achievement thatwill improve not only carrier testing but also the ability to measure the severity and progression of the disease.

"Formerly, mutations in this gene could be identified with the most common commercially available test in only about 60 percent of cases," said Sharon E. Hesterlee, Ph.D. "Dr. Flanigan's work opens the door for the other 40 percent of boys to receive accurate and affordable genetic diagnosis."

Parent Project Muscular Dystrophy, the Muscular Dystrophy Association, and the National Institutes of Health have funded the U researchers' work on MD.

Along with Flanigan and Weiss, other U of researchers on the project include Andrew von Niederhausern, Diane M. Dunn, and Jonathon Alder of the Utah Gene Depot. Jerry Mendell, M.D., of the Department of Neurology at Ohio State University, provided additional DNA samples for the Utah researchers to test.