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Fragile X Syndrome (Martin-Bell Syndrome) Amber Boone www.fragilex.org.uk/ page6.htm Characteristics Mild to Moderate Mental Retardation Long, narrow face Large, protuberant ears Macroorchidism (enlarged testicles) Background X-linked disease Mutation is located at Xq27.3 FMR1 Gene – Polymorphic (CCG)n repeat in the 5’ untranslated reagion of exon 1 – Hypermethylation of a CpG island upstream of the mutation Finding the Causitive Gene Cloned the X Chromosome from a normal human into YACs Digested with EcoRI – Found a 5 Kb region that was unstable in pedigrees with Fragile X (pfxa1) Digested with PSTI – Narrowed the instability down to a 1 Kb region (pfxa2) Sequenced this region Pfxa2 sequence Finding the Causitive Gene (cont) Used several RE to cut normal and Fragile X DNA isolated from human lymphnodes – Normal fragile X site varied from 45-95 bp – Infected individuals fragile X site was almost 900 bp longer Finding the Causitive Gene (Cont) Physical map across the Fragile X region – Mostly done through Resriction Enzyme cleavage Finding the Causitive Gene (cont) Isolated a YAC in somatic cell hyprids containing part of the Fragile X site Bought a YAC library and used their clone as a probe – Obtained a YAC with the whole region Created a cosmid library from the YAC clone Cosmid subclones used to screen a cDNA library of human fetal brain RNA – The cosmids hybridized to a portion of a gene designated as FMR1 Pedigree of a family with the Fragile X mutation segregating FMR1 Expression Northern Blot of FMR1 was done on Human Tissue – Expressed in highest levels in the Brain and Testes – Slightly lower level in the Placenta, Lungs, Liver, and Kidneys FMR1 expression was turned on early in embroyonic development Mouse Knockout More Findings Fragile X phenotype is caused from lack of FMRP expression FMRP was expressed in some men with full mutation, but no methylation Found alternatively spiced FMRP proteins in different locations in the body Treatment Study on in vitro reactivation – Rare cases of individuals with the full mutation and unmethylation have shown that the problem is in the methylation, which inhibits the translation – 5-azadeoxycytidine to induce DNA demethylation in vitro Bibliography Abitbol, M., Menini, C., Delezoide, A, Rhyner, T., Vekemans, M. and Mallet, Jacques. (1993) Nucleus basalis magnocellularis and hippocampus are the major sites of FMR-1 expression in the human fetal brain. Nature Genetics, 4: 147-152. Annemieke, J.M. et. al. (1991) Identification of a Gene (FMR1) containing a CGG Repeat coincident with a Breakpoint cluster Region Exhibiting Length. Cell, 65: 905-914. Bell, M. V., et al. (1991) Physical Mapping across the Fragile X: Hypermethylation andn Clinical Expression of the Fragile X Syndrome. Cell, 84: 861-866. Chiurazzi, P., et al., (1998) In vitro reactivation of the FMR1 gene involved in fragile X syndrome. Human Molecular Genetics, 7: 109-113. Dutch-Belgium Fragile X Consortium, (1994) Fmr1 Knockout Mice: A Model to Study Fragile X Mental Retardation. Cell, 78: 23-33. Froster-Iskenius, U., et al., (1984) Transmission of the marker X syndrome trait by unaffected males: Conclusions from studies of large families. Human Genetics, 67: 419-427. Hinds, H. L., et al., (1993) Tissue specific expression of FMR-1 provides evidence for a functional role in fragile X syndrome. Nature Genetics, 3: 36-44. Jin, Peng, et al., (2004) Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway. Nature Genetics, 7:113-117. Kirchgessner, C. U., et al., (1995) X inactivation of the FMR1 fragile X mental retardation gene. Journal of Medical Genetics, 32: 925-939. Kremer, E. J., et al., (1991) Mapping of DNA Instability at the Fragile X to a Trinucleotide Repeat Sequence p(CCG)n. Science, 252: 1711-1714. Mazroui, R., et al., (2002) Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression. Human Molecular Genetics, 11: 3007-3017. Ostra B. A., et al., (2001) The Fragile X gene and its function. Clinical Genetics, 60: 399-408. Sandberg, G., et al., (1997) Effect of in vitro promoter methylation and CGG repeat expansion on FMR1 expression. Nucleic Acids Resource, 25: 2883-2887. Thompson and Thompson, Genetics in Medicine 6th ed. The Curtis Center, Philidalphia, PA 2004 Verheij, C., et al., (1995) Characterization of FMR1 proteins isolated from different tissues. Human Molecular Genetics, 4: 895-901.
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