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Fragile X Syndrome (Martin-Bell Syndrome)

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

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Fragile X Syndrome (Martin-Bell Syndrome)

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  1. Fragile X Syndrome(Martin-Bell Syndrome) Amber Boone

  2. www.fragilex.org.uk/ page6.htm

  3. Characteristics • Mild to Moderate Mental Retardation • Long, narrow face • Large, protuberant ears • Macroorchidism (enlarged testicles)

  4. 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

  5. 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

  6. Pfxa2 sequence

  7. 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

  8. Finding the Causitive Gene (Cont) • Physical map across the Fragile X region • Mostly done through Resriction Enzyme cleavage

  9. 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

  10. Pedigree of a family with the Fragile X mutation segregating

  11. 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

  12. Mouse Knockout

  13. 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

  14. 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

  15. 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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