DIAGNOSIS OF MARFAN SYNDROME AT EARLIEST STAGE

August 10, 1995
Media Contact: Jo Martin
Phone: (410) 223-1736
E-mail: jmartin@welchlink.welch.jhu.edu

Researchers at the Johns Hopkins Children's Center have devised a method that should allow the reliable diagnosis of Marfan syndrome at the eight-cell stage of fetal development. Used in combination with in vitro fertilization, carriers of this inherited disease may now have the means to bear only unaffected, healthy children, they say.

Traditionally, scientists waited 10 to 12 weeks after conception to diagnose Marfan syndrome, says Hal Dietz, M.D., lead investigator of the study, which appears in the August issue of Nature Medicine. Because it can be severe, and pregnancy is taxing to women with the disorder, quick findings are especially important to carriers.

For the first time, Dietz and others effectively amplified RNA taken from an eight-celled fertilized egg not yet implanted in the womb. Their method allowed them to make many copies of genetic material -- enough to find a specific alteration in the genetic code.

In the past, this "preimplantation diagnosis" was used to detect other genetic disorders, like cystic fibrosis, by amplifying DNA. But that method proved unreliable for Marfan syndrome.

Marfan syndrome is an autosomal dominant, heritable disorder affecting connective tissue. Each offspring of a carrier has a 50-50 chance of inheriting the disease. It affects nearly one in every 10,000 people and is characterized by skeletal deformity, dislocation of the ocular lens of the eye, and expansion and ultimate rupture of the aorta, often resulting in death.

Hopkins scientist Victor A. McKusick, M.D., began establishing the clinical definitions of Marfan syndrome in the 1950s, but its molecular roots were not known until the late 1980s when Dietz and other researchers demonstrated that the primary defect was in the gene encoding fibrillin. Fibrillin is found in the body's connective tissue.

Mutations are permanent changes in DNA (deoxyribonucleic acid), where genetic information is stored. To find a mutation, researchers often "amplify" or make many copies of DNA using a technique called polymerase chain reaction or PCR.

During this process, the twisted ladder of DNA is split into two separate strands. A new half is rebuilt on each old half, then the ladder is split again and the process repeated over and over. Millions of copies of the DNA molecule that houses a gene are reproduced, providing enough material for scientists to quickly find a specific mutation.

To test PCR applicability for Marfan syndrome, Dietz and his colleagues studied single skin cells from a patient with a known mutation in the gene that encodes fibrillin. Problems arose, however. In contrast to expectations, the PCR amplification technique copied only one or the other of the two fibrillin genes present in any given cell. If only the normal gene copied, misdiagnosis of Marfan syndrome was likely, since only one of the two genes needs to be defective for the syndrome to appear.

"DNA amplification gave us only two copies of each fibrillin gene to start with," explains Dietz. "Because each gene makes many copies of RNA (ribonucleic acid), we asked whether PCR could be used to reliably amplify RNA."

Again using single skin cells from a Marfan patient, they showed not only that they could amplify RNA, but also that both the normal and altered genes were present.

Hereditary information flows from DNA to messenger RNA (mRNA). Millions of copies of the skin cell's mRNA were reproduced, forming a blueprint that allowed the researchers to work backward and rebuild the DNA sequence of the coding region of the fibrillin gene.

"We found consistent evidence of the mutated fibrillin gene," says Dietz. "But this information came from an adult. We still needed to know if an embryo, only eight-cells old, could produce the same information. It was a long-shot proposal," he says. "But it worked."

In conjunction with researchers from Cornell University's Center for Reproductive Medicine, the study was repeated with information from eight-celled embryos that were generated for other clinical indications, but were no longer viable for artificial implantation.

"We successfully amplified the RNA each time," says Dietz. "And we proved that this fibrillin RNA was derived from copies of the fibrillin gene that were inherited from both parents."

Because of that success, researchers are now prepared to offer this form of diagnosis to those who request it. A preimplantation diagnosis facility is currently being developed at Hopkins, says Dietz, and should be available in the near future. The Hopkins team is studying whether their technique for preimplantation diagnosis can be applied to other potentially fatal genetic disorders.

Other researchers are Zayd Eldadah, an M.D., Ph.D. student at the Johns Hopkins University School of Medicine, and Jamie Grifo, M.D., Ph.D., from the Center for Reproductive Medicine, Cornell University.


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