LONDON - A study of the genes that contribute to a rare inherited disorder has provided new insights into the control of mammalian development and could help to identify new strategies for controlling processes associated with cell growth and cancer.

Researchers at the University of Cambridge in Cambridge, UK, wanted to elucidate the roles and potential interaction of two genes known to be involved in Beckwith-Wiedemann syndrome. This rare overgrowth condition, which is inherited in about 15 percent of cases, is characterized by high birth weight and rapid weight gain after birth, as well as defects of the abdominal wall such as omphalocele, and an enlarged tongue. These patients have an increased incidence of childhood tumors.

The genes responsible for Beckwith-Wiedemann syndrome (BWS) map to the short arm of chromosome 11, to a region known to contain a cluster of imprinted genes. In contrast to most genes, imprinted genes are not expressed from both of their copies. They carry some kind of "imprint," the nature of which is not fully understood, which allows the cell to transcribe the genetic message from either the paternally derived copy of the gene or the maternally derived copy.

Many imprinted genes are involved in growth and development, and as a general rule those that are maternally expressed are growth inhibitors, that those that are paternally expressed are growth promoters.

The genes implicated in BWS are no exception. A gene known as IGF2 is expressed from the paternally derived copy of the gene, and is a growth promoter, while that known as CDKN1C p57kip2 is expressed from the maternally derived copy of the gene, and is a growth inhibitor. These genes encode proteins called insulin-like growth factor-II (IGF-II) and p57kip2. The latter is a cyclin-dependent kinase inhibitor.

Anne Ferguson-Smith, lecturer in the Department of Anatomy at the University of Cambridge, told BioWorld International: "The process of imprinting poses a number of questions. Why did this process of imprinting evolve, and what is the mechanism that allows the transcriptional machinery of the cell to tell the difference between the two seemingly identical chromosome homologues and only activate one and not the other? Furthermore, what are the functions of these imprinted genes and what are the clinical implications when imprinting goes wrong? One of the syndromes that has been attributed to abnormalities in the expression of imprinted genes is Beckwith-Wiedemann syndrome, which can help us answer some of these questions."

Ferguson-Smith and her team decided to investigate further and results are described in the May 9, 2000, Proceedings of the National Academy of Sciences in a paper titled "Increased IGF-II protein affects p57 kip2 expression in vivo and in vitro: Implications for Beckwith-Wiedemann syndrome."

They already knew from the body of research that there is loss of imprinting of the IGF2 gene in many cases of Beckwith-Wiedemann syndrome. In other words, whereas the maternal allele of IGF2 is normally inactive, in these cases both alleles are active so that levels of IGF-II are abnormally high. In some patients, the syndrome results because instead of inheriting one copy of the gene from each parent, two are inherited from the father and none from the mother.

Other studies examined the possible genetic causes of the familial Beckwith-Wiedemann cases, and found that about 5 percent of these had mutations in their p57kip2 gene. Ferguson-Smith said, "This gene, being maternally expressed and a negative regulator of growth, is a very different sort of gene to IGF2. The question is how defects in such different genes can result in the same syndrome. One possibility is that they are interacting in the same pathway and that overexpression of IGF2 might be affecting p57kip2 in some way."

Mouse models also provided clues to the function of IGF-II and p57kip2. Those that overexpress IGF2 have many (although not all) of the features of Beckwith-Wiedemann syndrome. Those that have mutations in p57kip2 also have some but not all of the features of the disease.

The publication of a paper describing a mouse model that contained even higher levels of IGF-II, which had more features of Beckwith-Wiedemann syndrome than those mice containing just a double dose of the gene, suggested to Ferguson-Smith that IGF2 may be affecting the animals' phenotype in a dose-dependent manner.

She said: "So we decided to do some simple experiments to see if IGF2 could affect expression of p57kip2 in a dose-dependent manner." Using a strain of mice with elevated circulating levels of IGF-II, they examined what was happening to p57kip2 expression during development. Ferguson-Smith said, "We could see from Northern blot analysis that p57kip2 was repressed by about 50 percent in the embryos of these mice. This indicated to us that perhaps there was some pathway in which IGF-II levels could influence p57kip2 expression."

Because they wanted to look at whether this effect was dose dependent, they also developed an in vitro model. They used primary embryonic fibroblast cultures, grew them in the absence of serum to ensure that no IGF-II was present, and then added different concentrations of IGF-II, before examining what was happening to p57kip2 expression.

Ferguson-Smith said, "We found that p57kip2 expression was down-regulated in a dose-dependent manner in response to IGF-II." Further tests showed that these effects were not caused by stimulation of cell division by IGF-II.

In the PNAS paper, the researchers conclude: "This down-regulation is not due to a relative increase in the number of actively dividing cells . . . but represents a direct or indirect effect of IGF-II via an IGF-II-related pathway."

Ferguson-Smith concluded: "This is very good evidence that IGF-II and p57kip2 act in the same pathway, at least in many of the developmental functions that are affected in Beckwith-Wiedemann syndrome. This finding has implications for development, for patients affected by this syndrome, and for cancer therapy as well."

She and her colleagues are currently looking for partners to help them develop this work. She added, "Next we want to address whether this interaction between IGF-II and p57kip2 is direct or indirect. We want to characterize the actual pathway involved, and ask what the relationship is between cell cycle control via p57kip2 and growth via IGF-II. We are also interested in the relative distribution of IGF2 and p57kip2 protein in the developing organism and whether this reflects the cell types and organs which are particularly susceptible to tumors or developmental growth defects."

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