1. Fragile X Syndrome (Martin-Bell Syndrome)
Amber Boone
I did my research on Fragile X Syndrome
First diagnosed in 1943 by J. Purdon Martin and Julie Bell
Studied a family with 11 severly retarded males and found that the inheritance pattern seemed to be X-linked
In 1969 an abnormality at the long arm of the X chromosome in 4 mentally retarded brothers and 2 of their mentally retarded female relatives was found. The long arm looked like it was broken or fragile hence the name Fragile X syndrome

2. www.fragilex.org.uk/ page6.htm
This image of the Fragile X chromosome made visible by atomic force microscopy
Arrow points to the “fragile” portion on the long arm of the X chromosome. As you can see it almost looks like its broken.
page6.htm

3. Characteristics Mild to Moderate Mental Retardation Long, narrow face
Large, protuberant ears
Macroorchidism (enlarged testicles)
Fragile X syndrome is one of the most common cause of inherited mental retardation. Statistics vary, but it is suggested that 1 in 1250 males and 1 in 2500 females are effected by it.

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
The papers use (CCG)n and (CGG)n interchangable. I’ll use (CCG)n in this discussion.
Fragile X Mental Retardation
This gene consists of 9 exons. The mutation occurs in exon 1, which is an untranslated region. Normal individuals have about 5-50 repeats. A carrier will have , and an infected individual usually has >200 repeats.
The number of repeats increases from generation to generation until an infected individual is produced. This process is called genetic anticipation.

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
E.J. Kremer, et al. June 21, 1991
The FMR1 gene as the cause of fragile X syndrome was discovered in 1991 by several teams of researcher.
The first step the researchers took was to determin the exact region of the mutation on the X-chromosome.

6. Pfxa2 sequence
In normal individuals this is what the fragement looks like.
Pfxa2 contained a CpG rich region (nucleotides 1 – 357) that contained seven RE sites.
It also contained a CCG repeat region that was about 120 nt long (nt 358 – 476). Contains about 40 repeats

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 bp
Infected individuals fragile X site was almost 900 bp longer
Tried to PCR ampliphy using oligodeoxyribonucleotide primers but this didn’t work. They could amplify the regions flanking the repeat but the repeat itself wasn’t stable. The clones were smaller than anticipated which indicated an in vitro deletion of the unstable region.
Previous sequencing demonstrated that this region was a (CCG)n repeat region
Couldn’t determine if the CCG region was amplified or if an unknown sequence was inserted into the repeat region and always lost during amplification
Assumed it was amplification of the repeat region because the region would progressively get larger generation after generation until an infected individual was found in the pedigree
Also found several Methylation sites in the pfxa2 region

8. Finding the Causitive Gene (Cont)
Physical map across the Fragile X region
Mostly done through Resriction Enzyme cleavage
M. V. Bell et al. and Annemiede J.M., et al.
Compare many different Southern Blots of DNA cut with varying numbers and types of RE.
-analyzed where each restriction site was compare to another
They found two clones flanking the fragile X site. They called these clones M759 and M749. Used parts of these clones as probes to pull out fragile X DNA from infected individuals.
Picture from Bell
Part A shows what a normal males RE fragments look like when cleaved with BSSHII
B shows that infected individuals give no band at 600 Kb or a reduced band when cut with BssHII
C shows the carrier mother and her infected sons
both sons show a reduced band
The presence of a reduced band in some infected individuals suggests that the BssHII fragment is not a deletion, but an alteration in methylation patterns because methylation can inhibit RE cutting
So from this research they determined a physical map with marders on both sides of the fragile X site. They also developed the idea that there was abnormal methylation.

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
They now had the area pretty well determined and they wanted to find a gene.
Annemiedi et al was the first group to find the gene.
They suspected there was a gene because the CpG island is a good indicator of a promotor in higher eukaryotic DNA. They suspected this gene could be inactivated by methylation
Fragile X Mental Retardation 1 (FMR1)
After characterization they found that exon 1 of FMR1 contained the repeat region and that the region was being expanded in fragile X syndrome pedigrees.

10. Pedigree of a family with the Fragile X mutation segregating
To show this here is a southern blot showing the segregation and expansion of the premutation. The grandmother shows very little mutation. The mother shows a larger premutation. And the son shows the full mutation. You can also see that the grandmother has a mildly effected son with the full mutation, however the mutation is not methylated and the son is not as severly affected. The picture also show an unaffected son with the normal allele.
Through different genetic studies researchers observed that the DNA plymerase paused at both hairpin and tetrahelical structures. This allowed for the formation of secondary structures on the lagging strand. This casued increase in rpeat length (up to 10 repeats).
The full mutation is encountered when an “expansion threshhold” is reached (occurs around 70 repeats). Multiple hairpins and stem and loop structures form on the lagging strand. These slip at both ends and are unstable. This results in a variety of expanded full mutations. Fragile X patients are often mosaics for full mutations of different sizes. One theory is that the abnormal structures attract de novo methyltransferases which methylate the whole CpG island in an attempt to stop the expansion. This causes the silencing of the FMR1 gene. However there is other research that indicates methylation is caused from incomplete X reactivation in the female germ line. This is supported by inheritance patterns. Full mutations are only spread maternally. The mutations are actually shortened when passed paternally. This is new research and there is not much known yet.
But, to get back to this, sequencing analyses also showed that the CGG repeats were interupted by AGG triplets. These triplets seem to help stabalize the gene and prevent replication slippage. It’s been found that the loss of the most distal 3’-AGG is an important determinant in CGG instability.

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
Now they knew what gene was involved and that fragile X syndrome was caused by the lack of FMR1 expression. They wanted to find out what FMR1 does.
To do this studies were done on FMR1 expression levels in different tissues and different stages of development were tested.
Shows that it is expressed in highest levels in the Brain and Testes, and in lower levels in the Placenta, Lung, Liver and Kidneys
In situ hybridization was used to show embroyonic expression. This was done in mice because mice FMR1 have a 95% identity to human FMR1

12. Mouse Knockout
After learning what tissues FMRP is expressed in and kind of when it is turned on in empryonic development the next step was to create a mouse knockout.
This procedure was interesting because the procedure they used was exactly the same as we learned in class.
The first picture shows a schematic view of what the FMR1 gene looked like and the bottom part of the picture shows where they inserted the neomycin and thymidine kinase marker genes.
The second picture shows a PCR analysis of the mutation. Lane one is from ES clones. Lane two is from a wild type mouse, lane three and four is a heterozygous female. 5 and 6 is from mutant male. 7 and 8 are from mutant females.
This showed that all the infected individuals had the 900 bp allele. The heterozygous individuals had a 900 bp allele and a 420 bp allele. The unaffected individuals only had the smaller allele. They used this method to identify the knockout allele in the female mice.
Next they tested the phenotypes of these mice. The physical phenotype of testicular weight with age was measure. The results are shown in the third figure. The weight of the mutant mice was significantly larger than the control mice and the size difference increased with age.
They also did several behavioral and motor activity tests on the mice. They concluded that the mice showed the same phenotype as human Fragile X patience showed and that the mouse was a good animal model for the disease.

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
One form of the FMRP is involved in RNA binding. Researchers believe that the FMR1 protein acts as a shuttle within cells carrying mRNA from the nucleus to areas of the cell where proteins are assembled.
Also, proteins interacting with FMRP have been identified, and suggest a link with the Rac1 GTPase pathway that is important in neuronal maturation
The FMR1 protein may help regulate synaptic plasticity, which is important for learning and memory.
So, in general research has shown many different areas of expression and many possible purposes for the FMRP. More research to find out all the purposes of FMRP is currently underway.

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
The picture shows that it is possible to unmethylate the gene in vitro. This is RT-PCR products of FMR1 and HPRT with and without 5-azadC treatment. 2,4,6,8, and 10 are untreated cells. Twelve is an untreated control males and thirteen is a treated control male.
This could possible lead to a preventative treatment.

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:
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:
Bell, M. V., et al. (1991) Physical Mapping across the Fragile X: Hypermethylation andn Clinical Expression of the Fragile X Syndrome. Cell, 84:
Chiurazzi, P., et al., (1998) In vitro reactivation of the FMR1 gene involved in fragile X syndrome. Human Molecular Genetics, 7:
Dutch-Belgium Fragile X Consortium, (1994) Fmr1 Knockout Mice: A Model to Study Fragile X Mental Retardation. Cell, 78:
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:
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:
Jin, Peng, et al., (2004) Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway. Nature Genetics, 7:
Kirchgessner, C. U., et al., (1995) X inactivation of the FMR1 fragile X mental retardation gene. Journal of Medical Genetics, 32:
Kremer, E. J., et al., (1991) Mapping of DNA Instability at the Fragile X to a Trinucleotide Repeat Sequence p(CCG)n. Science, 252:
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:
Ostra B. A., et al., (2001) The Fragile X gene and its function. Clinical Genetics, 60:
Sandberg, G., et al., (1997) Effect of in vitro promoter methylation and CGG repeat expansion on FMR1 expression. Nucleic Acids Resource, 25:
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:
Tons of research done on this topic. Hard to sort through the data, because of conflicting data.