June 13, 1998 A gene implicated in hydrocephalus and a number of other birth defects in mice has been discovered by scientists at Vanderbilt University Medical Center, the researchers report in the June 12 issue of the journal Cell.
Working with colleagues in Canada, the researchers have linked mutations in the corresponding human gene to glaucoma, a leading cause of blindness in the United States. The gene, called mouse forkhead 1 (mf1), is also associated with defects in the skeleton, the cardiovascular system and kidneys.
"The gene that we cloned is almost identical in mice and humans," said Brigid L.M. Hogan, Ph.D., Howard Hughes Medical Institute investigator and Hortense B. Ingram Professor of Molecular Oncology. "We are excited because the mutant mice can now be used to understand organ systems in the human."
With roots in a classic scientific finding of more than 50 years ago, the discovery offers a clear example of how basic research may ultimately be connected to human disease in ways that could not be imagined at its beginning.
The new findings build upon the research of Hans Gruneberg, a German-born scientist working in the United Kingdom. In 1943, he was the first to describe a spontaneous mouse mutant known as congenital hydrocephalus (ch). These mutant mice, which died soon after birth, were born with a large swelling of the brain that is filled with cerebrospinal fluid (hydrocephalus). They also have several other abnormalities, including open eyes -- they are usually closed in newborn mice -- a missing sternum and other skeletal defects, and problems with their blood vessels and kidneys.
In the 1970s, scientist Margaret Green, working at the Jackson Laboratory in Bar Harbor, Maine, studied the ch mutant mice and described a malformation in the meninges, the spongy tissue around the brain. This defect prevented the proper flow of the cerebrospinal fluid out of the brain through the meninges and into the bloodstream. Since then, the ch mutant strain has been kept in the Jackson Laboratory, awaiting future researchers to identify the genes responsible for these abnormalities.
Meanwhile, scientists in Hogan's laboratory at Vanderbilt were working with genes important in the embryonic development of flies. They were searching for corresponding genes in mice, which, in turn, offer a good model for studying genes in humans. They had zeroed in on a fly gene called forkhead, which when mutated results in larvae whose heads do not develop properly.
"We began looking in mice for genes that are related to the fly forkhead gene, and we found a number of them," Hogan said. "There are probably about 50 members of this family, but we started focusing on one of them, which we called mouse forkhead 1."
Hogan's team cloned the gene and then engineered special "knock-out" mice that lack this gene completely. When the mice without the mf1 gene were born, they exhibited a long list of abnormalities: hydrocephalus, a missing sternum, spina bifida, open eyes, thickened corneas, under-developed irises, vascular defects, and kidney problems.
The researchers immediately made the connection to the classic ch mutant and obtained mice from the Jackson Laboratory to test their suspicions. By cross-breeding the Jackson mice with their mf1 knock-out mice, they determined that the same gene was involved in both sets of abnormalities
When Hogan's group sequenced the mf1 gene from the ch mutant mice, they found that instead of producing the full protein, it stopped short, resulting in an incomplete protein that doesn't work properly.
"We'd made our mutant by knocking out the gene completely, but nature had made this mutation spontaneously," Hogan said. "The ch mutant has the gene, but a single nucleotide change in its DNA sequence alters the message so that it stops short."
The specific link to glaucoma came after geneticists at the University of Alberta heard about the work being done in Hogan's lab and contacted the researchers in Nashville.
Led by Michael Walter, a co-author on the Cell article, the Canadian team was studying families with an inherited defect in the iris (iridogoniodysgenesis type 1 or IRID1) that puts affected children at high risk of developing glaucoma. Glaucoma is characterized by damage to the optic nerve, believed to result from the pressure of excessive fluid building up inside the eyeball.
The Canadian group had identified a candidate human gene, FREAC3, which turned out to be almost identical to the mouse mf1.
The build-up of fluid in the eye in glaucoma may be similar to the accumulation of cerebrospinal fluid in the brain of the mf1 knock-out mice, Hogan noted. In patients with IRID1, the fine meshwork of cells in the iris through which the ocular fluid normally drains does not develop properly, Hogan said.
"Our gene is expressed in this meshwork of cells, and we observed obvious eye abnormalities in our homozygous knock-out mice," Hogan said.
The two groups of investigators began exchanging information. VUMC researchers shared the sequence of the mouse gene, and the Canadian researchers have subsequently identified mutations in the FREAC3 gene in several families and patients with the eye disease. Working independently, another group of researchers at the University of Iowa recently found other similar mutations in affected families.
Each mouse or person has two copies of each gene. The human FREAC3 mutation appears to be dominant, meaning that only one "bad copy" of the gene is required for an increased susceptibility to glaucoma, Hogan said. "Controlling the balance of fluid in the eye may be very sensitive to relatively small changes in the behavior of the meshwork cells," she said.
The heterozygous mf1 knock-out mice -- those with one working copy of the mf1 gene -- did not exhibit any obviously severe eye disorders. However, the researchers are now collaborating with Simon John, Ph.D., at the Jackson Laboratory, who can measure eye pressures in the mice to determine whether subtle abnormalities exist.
"We're interested in seeing whether certain abnormalities are more or less severe depending on the genetic strain of mice," Hogan said. "The mf1 gene is a transcription factor, which means that it acts by switching on other genes in response to signals to the cell. The interaction of different genes in different genetic backgrounds may help explain why some members of a family who inherit mutations are more likely to develop symptoms while others do not."
The work may also have future implications for treatment of glaucoma.
"This gene may be only a part of a jigsaw puzzle," she said. "Eventually, if we can understand more about how this gene works and how these abnormalities develop, the research may lead to the development of new drugs to counteract glaucoma."
The research was funded by the Howard Hughes Medical Institute. In addition to Walter, Hogan's collaborators include post-doctoral researchers Hiroshi Sasaki and Tsutomu Kume; Ke-Yu Deng and Virginia Winfrey of the Vanderbilt Department of Cell Biology; and University of Alberta researcher Douglas B. Gould.
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