Reactivation of the FMR1 Gene in Fragile X Patients Cells in Culture

With a $62,000 grant from FRAXA Research Foundation from 1999-2000, Dr. Giovanni Neri and his team at Universita Cattolica del S. Cuore explored  possible strategies to turn the Fragile X gene back on.

$62,000 Grant

Giovanni Neri, PhD
Principal Investigator

Universita Cattolica del S. Cuore
1999-2000 FRAXA Research Grant
$62,000 over 2 Years

FRAXA funded this work during 1999-2000. In individuals with Fragile X syndrome a chemical switch called methylation “turns off” the FMR1 gene. Simple molecules known as methyl groups attach to the DNA sequence constituting the “promoter” region of the gene, effectively blocking the transcription of its genetic code. The result is gene inactivation and lack of its specific protein product FMRP.

In addition, proteins known as histones make up a spool around which the DNA thread is wound. If the histones are loaded with acetyl groups (again, simple modifying molecules like methyl groups), the DNA is loosely packed and the FMR1 gene can be freely transcribed. On the other hand, loss of acetyl groups (deacetylation) results in tight packing of the DNA, which becomes inaccessible to the molecular machinery responsible for the transcription of the gene and, ultimately, for the production of FMRP. Thus, DNA hypermethylation and histone deacetylation need to be reversed to enable the FMR1 gene to express FMRP.

by Giovanni Neri, 1/30/1999

Our project is based on the fact that in 99% of the Fragile X syndrome patients, the coding region of the FMR1 gene is intact and its loss of function is due to the CGG expansion and methylation of the regulatory region at one of its extremities. One can say, in a simplified manner, that the gene is switched off and that we are trying to find a way to switch it back on.

Since methylation is a key factor in the silencing of the gene, our first approach was to treat in vitro cell lines from Fragile X patients with the demethylating drug 5-azadeoxycytidine. This treatment reactivated FMR1 gene transcription and restored production of its protein product FMRP (Chiurazzi et al., Hum Mol Genet 7, 109-113, 1998). We plan on extending our original observations to a larger number of cell lines and at the same time on trying other compounds that have the potential to reactivate the FMR1 gene.

One promising compound is butyrate, which acts on DNA-associated proteins called histones. Histones play an important role in the regulation of gene activity and in the past we were able to show that treating Fragile X lymphocytes (blood cells) with butyrate resulted in a reduced manifestation of the fragile site FRAXA (Pomponi and Neri, Am J Med Genet 51, 447-450, 1994). Butyrate is a drug of limited toxicity and the results obtained with the in vitro experiments could lead to eventual in vivo trials (in live animals or human patients.) International collaboration with other distinguished groups (Ben A. Oostra, Rotterdam; Steve Warren, Atlanta) will allow the sharing of expertise and will help in developing new ideas and new approaches.

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