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Reverse Genetics Methods: What's Known, What's New, What's Next On The Agenda

The last decade has seen extraordinary advances in identifying single genes that are responsible for a variety of human diseases. Most recantly researchers have pinpointed the genes that cause retinoblastoma, chronic granulomatous disease, muscular dystrophy, and cystic fibrosis, the last as recently as September. Ingenious experimental design and new technologies are helping to pave the way for rapid identification of other disease-causing genes. And from there, the development of improved d

By | November 27, 1989

The last decade has seen extraordinary advances in identifying single genes that are responsible for a variety of human diseases. Most recantly researchers have pinpointed the genes that cause retinoblastoma, chronic granulomatous disease, muscular dystrophy, and cystic fibrosis, the last as recently as September. Ingenious experimental design and new technologies are helping to pave the way for rapid identification of other disease-causing genes. And from there, the development of improved diagnostic procedures and therapeutic treatments is sure to follow, say experts.

For most genetic diseases, the normal function of the involved gene is not known. Localizing a disease-causing gene without knowing anything about the disease’s molecular or biochemical nature is , called “reverse genetics.” The process of identifying such genes is laborious, since there are between 50,000 and 100,000 genes distributed within 3 billion base pairs of DNA. But recently developed techniques are greatly expediting such searches for the proverbial “needle in the haystack.”

RFLP

One technique is restriction fragment length polymorphism (RFLP), first described in 1980 by David Bots’tein of the Massachusetts Institute of Technology (D. Botstein, American Journal of Human Genetics, 32:314-31, 1980). Briefly, RFLPs are variations in the DNA that can beidentified after the DNA is cut by restriction enzymes. These RFLPs act as markers that can be linked to genes responsible for certain diseases.

But to use RFLPs, “a family study is necessary to determine if a DNA variant exists near the disease gene under study,” says Clay Stephens of the Human Gene Mapping Library in New Haven, Conn. If no family members have the variant or if the disease is due to a new mutation, RFLPs cannot be used.

Yet under the right circumstances, RFLPs are an invaluable ge "netic tool, as James F. Gusella of the Massachusetts General Hospital in Boston found in his hunt for the general chromosomal location of the gene causing Huntington’s disease. In 1983 Gusella and colleagues used RFLPs to confirm that the gene for this disease is on chromosome 4 (J.F. Gusella, et al., Nature, 306:234-8, 1983).

Gusella is one of the most cited authors in the Huntington’s disease ‘research area. His Nature paper is one of 10 that are “core” papers in the Science Citation Index-derived 1988 research front, or specialty area, on Huntington’s disease. This specialty contains some 220 papers published in 1988 that consistently cited one or more of the 10 core papers.

Chromosomal Jumping

In addition to RFLP markers, another technique that has aided identification of disease-causing genes is chromosomal jumping, developed in the laboratory of Francis C. Collins at the University of Michigan Medical Center in Ann Arbor. Using this method, researchers scan chromosome segments quickly, jumping from gene to gene, five to 10 times faster than with conventional methods. In a paper published in September, Collins and colleagues reported using chromosome jumping to help locate the cystic fibrosis gene (J.M. Rommens, et al., Science, 245:1059- 65, 1989).

One of the first successes of reverse genetics was achieved by researchers of Duchenne Muscular Dystrophy (DMD). In 1985 Louis M. Kunkel and colleagues of Children’s Hospital in Boston were able to isolate DNA fragments that were missing from a patient’s X chromosome (L.M. Kunkel, Proceedings of the National Academy of Sciences USA, 82:4778-82, 1985). Eventually, Kunkel’s team linked these DNA segments to the defective gene causing DMD (M. Koenig, et a1., Cell, 50:50947, 1987).

These papers are two of the 27 core articles in ISI's 1988 research front on DMD. This specialty contains 421 papers published in 1988 that referred to the 27 core articles. A statistical analysis of the citing author addresses listed on the 421 papers revealed that U.S. scientists contributed more than 45% of the literature in this area. The table on page 18 lists the institutions that are most actively pursuing further work on this X-linked disease. The number beside each institution indicates its percentage contribution to the 421 papers.

What’s Next?

According to Stephen T. Reeders of Yale University, researchers are coming very close to isolating the gene causing adult polycystic kidney disease (APKD), the third most common cause of kidney failure and a disorder that strikes 1 in 1,000 peopIe in the United States. So far the location of the APKD gene has been narrowed to a small region on the short arm of chromosome 16 (S.T. Reeders, Genomics, 3:150-5, 1988.)

. To find this narrow chromosome region, investigators used the same techniques implemented in hunting for the DMD and cystic fibrosis genes. But identifying the gene causing APKD will require different strategies, says Reeders. For instance, DMD occurs when a portion of the X chromosome containing the DMD gene is deleted. “No deletion has been found in APKD cases that would pinpoint the disease’s gene location,” says Reeders. And when researchers isolated a small chromosomal region that contained the cystic fibrosis gene, only one or two genes were located in that region. But the APKD chromosomal region contains many genes that must be sorted through. Daunting as this gene hunt may seem, Reeders says, “I would be disappointed if the APKD gene isn’t isolated within the next two years.”

Also very close to being isolated are the genes that cause Huntington’s disease and neurofibromatosis. Stephens says, it won’t be long before all the genes causing single-gene disorders—some 4,000—will he identified.

“The next step in gene identification,” Stephens adds, “will probably focus on the more complicated problem of multifactorial disorders, such as heart disease, which are caused by a combination of multiple genetic problems and environmental factors.”

Implications

The most immediate benefit of identifying disease-causing genes is improved diagnosis. Physicians will have more reliable tests at their disposal for determining whether disease is present or will develop in a ferns, newborn, or adult and whether that defect might be passed on to offspring.

But perhaps the ultimate goal of identifying disease-causing genes will be to implant copies of “healthy” genes into the body and get them to a majority of cells to treat or repair genetic disorders, which will mark the dawn of the brave new world of gene therapy.

Lisa Holland is a freelance writer based in New York City. Ricki Lewis s teaches biology at the State University of New York, Albany.

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