In humans, the disorder stems from a mutation on the X chromosome as a three-base sequence begins to repeat over and over in a section of the fragile X mental retardation 1 gene (FMR1). The portion of the gene where this error multiplies does not code for a protein, which means that several repetitions of the sequence can occur without damaging the fragile X mental retardation protein (FMRP). People who have a gene with a sequence that is repeated 50 or fewer times are considered normal; those with fewer than 200 repetitions are carriers of the disorder. Individuals with more than 200 triplets, however, have disruptions to the promoter region of FMR1 that block the gene from being transcribed into RNA and forming a protein, thereby prompting onset of the syndrome.
Scientists had a tough time studying this process in mice, because the repeated sequence does not accumulate the same way in rodents. Hence, they could not determine the action that halted FMRP production, causing disorders from anxiety to attention deficit disorder as well as cognitive difficulties ranging from learning disabilities to mental retardation. The Israeli team reports in Cell Stem Cell how its model helped determine the process in which FMR1 is silenced.
"Human embryonic stem cells should not be considered only as sources in transplantation medicine; they can be used also to create models for human genetic disorders," says study co-author Nissim Benvenisty, a geneticist at The Hebrew University of Jerusalem. "This is the first example where we in this field learn something new about a human genetic disorder that we couldn't learn from the existing models."
Using embryos from a female carrier (who had 170 triplets on her FMR1 gene), the researchers created a stem cell line that developed a mutation severe enough to be consistent with fragile X. They implanted this cell line into a mouse with a severely suppressed immune system, which allowed it to proliferate into a teratoma—a tumor composed of cells that can form varying tissue types. The researchers then placed the cells in a lab culture, where they could be monitored as they began to differentiate.
The researchers observed that the FMR1 gene remained active and FMRP was produced before the cells differentiated. After that point, however, they saw some epigenetic effects (influences on the activity of a gene that are not due directly to DNA mutations). As differentiation progressed, the scientists noted that the chromatins (DNA chemical complexes) in the cells' nuclei were structurally modified, effectively silencing the FMR1 gene. "It's going from an open conformation where it is transcribed [into RNA and then translated into protein] to a closed conformation where it is not transcribed," Benvenisty says.
He adds: the gene becomes methylated—a process in which a bulky methyl molecule is added to a gene's DNA backbone, blocking it from being transcribed into a protein. This process offers a sort of maintenance of the inactive state.
FMR1 inactivation "is a unique example in which epigenetic modification is in response to genomic modification," Stephen Warren, a human geneticist at Emory University in Atlanta wrote in an editorial that accompanied the study.
Karen Usdin, a senior investigator who works on fragile X syndrome at the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, Md., says this is the model she would have wanted to develop for the illness. (She explains that restrictions on embryonic stem cell research, in place since 2001, prevent federal funding being used to generate new embryonic stem cell lines including those with specific disease mutations.) "It does create a wonderful model system," she says. "It allows you to begin to dissect out the process of gene silencing and test drugs that can reverse that process."
"What we are trying to do now is prevent the silencing," says Benvenisty. The Israeli team plans to study different drugs that hold the promise of preventing the conformational change that shuts off FMR1. Once they home in on potential candidates, Benvenisty says, researchers will have to determine if they are effective enough "to reverse the silencing when [the gene is] methylated."