Fragile X syndrome, which is one of the most common forms of inherited mental retardation, has been shown to result from mutation of the FMR1 locus at Xq27.3. The most frequent mechanism of mutation is expansion of a polymorphic CGG sequence located in the 5'-untranslated region of the gene past a threshold of about 200 repeats; this sequence is normally heavily methylated, which causes transcriptonal inactivation. The protein encoded by the FMR1 locus (FMRP) has been found in large ribonucleoprotein particles in the cell and it appears to specifically interact with a subset of mRNA species on translating polyribosomes; loss of functional protein then may result in translation dysregulation of these downstream genes. Another intriguing property of the locus in patient cell lines is delayed replication of a chromatin domain 10-20 times the size of the gene itself, which is likely to play a role in the characteristic observation of a """"""""fragile"""""""" site at this position. The investigators have made the surprising observation that this late replication phenotype is also found in cell lines with non-expansion mutations, including a large deletion in the 5' region, a single base deletion that leads to a frameshift and premature termination and a splice site mutation. The simplest explanation of this finding is that the FMRP protein is involved directly or indirectly (through chromatin modification, for example) in regulation of replication properties at this locus, and possibly others. The goal of this application is to test this hypothesis. To accomplish this, the investigators will use a fluorescence in situ hybridization technique to examine replication timing in additional cell lines from patients with non-expansion mutations, including a missense mutation that affects one of the primary RNA binding domains and microdeletions affecting gene expression. In addition, they will examine replication in cell lines from """"""""high-functioning"""""""" males, where it is postulated that a deficiency in methylation allows functional protein production in spite of a full expansion of CGG nucleotides past the normal level of affected males. The investigators will also examine directly the role of FMR1 in replication control by either activating endogenous inactive genes or by attempted complementation of the late replication phenotype using transfection with tetracycline-inducible cDNA constructs or genomic BAC clones. Finally, the investigators wish to conduct some preliminary experiments to study potential replication phenotypes elsewhere in the human genome as well as in mouse cells. They feel these studies represent a novel approach to study the possible functional roles of FMRP within the cell which may, in turn, lead to a major new direction in the study of the etiology of fragile X syndrome.
