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The Molecular Basis of Fragile X Syndrome

Page history last edited by Ben Johnson 14 years, 5 months ago

Introduction:

Fragile X syndrome is the common form of inherited mental retardation and is predominantly caused by a large expansion of CGG triplet repeats located in the 5'-untranslated region of the fragile X mental retardation-1 (FMR1) gene. (Jin et al. 2000). 

An estimated incidence of 1 in 4000 males and 1 in 8000 females is associated with Fragile X syndrome.

Transmission of the syndrome occurs via an X-linked dominant trait with reduced penetrance; being 80% in males and 30% in females. (Jin et al. 2000).

The clinical presentation of the fragile X syndrome is expressed by mild to severe mental retardation, with an IQ between 20 and 60. (Pandey et al. 2004).  

In 1969 a landmark discovery was made upon assessment of a marker X chromosome in a family with four males affected with mental retardation. A constriction site was observed at the end of the long arm of the X chromosome in all affected males and two females. This was the significant discovery that led to the development of the diagnostic method for the fragile X syndrome. (Pandey et al. 2004).  

Itwas in 1991 that the molecular basis of the syndrome was revealed by conducting positioning cloning techniques. This was one of the first identified human disorders caused by a dynamic mutation, trinucleotide repeat expansion. (Jin et al. 2000).     

 

Why is the Syndrome Termed 'fragile X'?

In patients with fragile X syndrome, it is usually possible to observe an unusual characteristic at the end of the X chromosomes when examined under a high powered microscope. This feature at the end of the X chromosome appears narrow and is known as the 'fragile site' (FRAXA). (Barlow-Stewart et al. 2007). 

This FRAXA site is located at Xq27.3 near the end of the long arm of the X chromosome. (Jin et al. 2000). 

 

Figure 1 - Comparison Between a Normal X Chromosome and a Fragile X Chromosome (extracted from 'Embryology', UNSW, 2009)

Figure 1 demonstrates the difference between a 'normal' X chromosome and a fragile X affected chromosome. A bulb-like feature can be noted at the end of the long arm of the fragile X chromosome.

 

Clinical Features:

The fragile X syndrome is a relatively subtle dysmorphic syndrome and can therefore be difficult to diagnose on a clinical level. The common clinical features of the syndrome include a long face with prominent mandible, large and mildly dysmorphic ears and macroorchidism (abnormally large testes). The phenotype is subtle in young patients but the features become more distinct with age. (Pandey et al. 2004).

In males, mental retardation (MR) varies from mild to profound with most affected males being moderately to severely retarded; whereas females are commonly less severely affected than males. (Pandey et al. 2004).  

Slight abnormalties in connective tissue, hyperactive and attention deficit disorder and autistic-like behaviour are also associated with many cases of fragile X syndrome. (Jin et al. 2000).

Figure 2 - Comparison of the Clinical Features of Fragile X Syndrome at Childhood and Adult (extracted from 'Fragile X Syndrome Blog', 2009)

Figure 2 demonstrates two patients with fragile X syndrome. The physical characteristics of the syndrome do not become noticeable until adulthood. These characteristics are highly subtle in childhood.  

 

Genetics Behind the Fragile X Syndrome:

The length of the CGG repeat sequences can be refferred to as short, medium and long. Most people have a short repeat length. Individuals with a medium repeat size have a working copy of the gene but are just carriers of the premutation and do not express symptoms of fragile X syndrome. Premutation carriers are not affected intellectually but are prone to developing a neurological condition after about 50 years of age. (Barlow-Stewart et al. 2007).

The long repeat size of the CGG repeat is known as the full mutation. Males with a full mutation have fragile X syndrome. Females with the full mutation in just one of their FMR-1 gene copies (and with a working copy on the other partner X chromosome) are carriers of the syndrome and may only be mildly affected with fragile X syndrome. 

The pattern of inheritance in families consisting of the faulty fragile X gene, is reffered to as X-linked recessive inheritance. When inherited from the mother, the size of the CGG repeats can increase. For example, a mother with a medium sized repeat (premutation) can produce a child with long repeat size in the FMR-1 gene copies (full mutation). (Barlow-Stewart et al. 2007).        

 

The Molecular Basis of the Fragile X Syndrome - Instability and Methylation of the CGG Repeats:

The fragile X syndrome is predominantly linked to the unstable expansion of a CGG repeat located in the 5' UTR of the fragile X mental retardation gene 1 (FMR-1). This generates an abnormal methylation pattern that frequently causes the transcriptional silencing of the gene. Rare atypical cases of fragile X syndrome have been reported in the past, whereby the syndrome has not been associated with an amplification of the trinucleotide repeat, but rather with deletions or single point mutations. (Zalfa et al. 2003).

Fragile X mutations have been classified as premutations. This classification is based on the small expansions of 50-200 copies of the unmethylated CGG repeat and full mutations ranging in size above 200 repeats, consisting of hypermethylation of the proximal CpG island and the expanded CGG repeat. (Pandey et al. 2004). 

Premutations are unstable and have been shown to expand to a full mutation in offspring with existing irregular methylation only after it has been transmitted by a female (maternal transmission). Another factor involved with the instability of the CGG repeat expansion, is its size. If the premutation is large, then it is highly unstable upon transmission. Normal sized CGG trinucleotide repeat alleles behave like other microsatellite markers and are stable upon transmission. (Pandey et al. 2004).

Past research involving the DNA sequencing of normal and premutation FMR-1 alleles have highlighted that the number and position of AGG interruptions within the CGG repeat sequence appears to be a significant instability determinant involved in the molecular mechanism of the syndrome. Normal alleles commonly consist of one or more regularly spaced AGG sequences whereby the uninterrupted CGG tracts are limited to no more than 30 repeats. Premutation alleles, however, have no (or at most, one) interspersing AGG triplet and a CGG copy number that exceeds 30 repeats at the 3' end of the repeat array. (Pandey et al. 2004).

It has been thought that the AGG interspersions are responsible for producing stability and that there absence results in the generation of longer CGG arrays with poor stability and increased predisposition to expansion. (Pandey et al. 2004). This model was unfortunately limited as it was not able to explain how slippage could produce the large expansion during transmission from premutation to full mutation. (Jin et al. 2000).

The methylation of CGG repeats was shown to silence transcription via histone deacetylation. The mechanism of FMR1 de-activation is characterised by the methylation of the expanded CGG repeats. A methyl-binding domain protein (MeCP2) binds to the hypermethylated CGG repeats and forms a complex with histone deacetylases (HDACs) via the transcription repressor (Sin3). The HDACs then deacetylate the H3 and H4 histones of chromatin around CGG repeats. This histone deacetylation results in chromatin remodelling (chromatin condensation) and represses transcription. Therefore, the fragile X mental retardation protein (FMRP) is not expressed and manifestation of the fragile X syndrome occurs. (Jin et al. 2000).  

 

Figure 3 - FMR1 Transcriptional Suppression (extracted from Jin et al. Oxford Journals, 2000)

Figure 3 demonstrates the mechanism of FMR1 transcriptional supression through the expansion of CGG repeats. The first (top) structure represents normal chromatin structure around CGG repeats. Step A shows the methylation of the (800) expanded CGG repeats. A methyl-binding domain protein, MeCP2 binds to the hypermethylated CGG repeats and forms a complex with histone deacetylases (HDACs) through the activity of the Sin3 transcription repressor (Step B). In step C, the HDACs deacetylate the H3 and H4 histones around CGG repeats. This histone deacetylation leads to chromatin remodelling (chromatin condensation) and transcription is supressed (Step D).   

 

The Molecular Basis of the Fragile X Syndrome - Absence of the FMRP Protein:

 

The clinical features of the fragile X syndrome are generated due to the absence of a set of protein isoforms derived from alternative splicing of the FMR-1 gene and are termed FMRP. FMRP is an RNA binding protein that is also part of a ribonucleoprotein particle involved in the active translation of polyribosomes. (Bardoni et al. 2001).

FMRP has been identified to act as a translational repressor of specific mRNA's at synapses. Alternatively, the FMRP can associate with the dendritic, non-translatable RNA BC1BC1 has the ability to bind directly to FMRP and can also interact with the mRNAs regulated by FMRP, in the absence of any protein. Hence, when FMRP is absent, specific mRNAs at synapses undergo loss of translational repression. This produces the fragile X syndrome. (Zalfa et al. 2003).

It has recently been elucidated that the FMRP interacts with large numbers of mRNA. Without FMRP, the mRNAs become misregulated and can then result in the mental retardation of fragile X. This occurs because important processes involving important neuronal functions, such as vesicle transport and signal transduction, are unable to function appropriately without the activity of the FMRP. (Pandey et al. 2004).

Knockout of the FMR-1 gene has been conducted successfully in mice in past experiments by utilising gene targeting. The mutant mice were shown to exhibit macroorchidism and a slight deficit in learning and memory capability, thereby modelling the symptoms of the human fragile X phenotype. (Jin et al. 2000).

The FMRP protein consists of two KH motifs and one cluster of arginine and glycine residues, known as the RGG box. This domain structure permits the FMRP to bind RNA homopolymers and mRNAs in vitro. (Zalfa et al. 2003). 

There are two forms of FMRP homologs in mammalian organisms - FXR1P and FXR2P. These are believed to have distinct but overlapping functions. (Zalfa et al. 2003).

Studies of RNA-binding specificity of FMRP have higlighted that FMRP specifically binds to poly(G) and poly(U) in vitro. Furthermore, FMRP can only bind to selective brain mRNAs in vitro, such as the 3'-UTRs of myelin basic protein (MBP) and the FMR1 message itself. (Jin et al. 2000).

Only the first KH domain of FMRP has been identified to bind to RNA. This experimental result has been shown to be consistent with the finding that FMRP, with a point mutation (I304N) at the second KH domain, can still bind to RNA. It has been hypothesised that FMRP is a protein consisting of various interaction points for RNA binding, whereby the N-terminus region provides sequence specificity and non-specific binding at the C-terminus. (Jin et al. 2000).  

The predominant phenotype of fragile X syndrome, mental retardation, is known to occur in the CNS and the 'normal' function of FMRP has been speculated. This condures the intriguing question - how does the absence of FMRP lead to cognition deficit?

Past research have provided overwhelming evidence that FMRP in the neurons, are likely to play an active role in the transportation of target mRNAs from the nucleus to the cytoplasm, aswell as mediate the localization of these mRNAs and further regulate the localised synthesis of proteins by forming with polyribosomes. These processes are considered to be crucial for normal neuronal development and associated functions. (Jin et al. 2000). 

One specific example that reflects the importance of FMRP is its involvement in the regulation of protein synthesis within the dendritic spine. This is essential for synaptic development and brain plasticity. Consistent with this concept is the fact that abnormal dendrtitic spines have been observed in the brains of fragile X patients and in FMR1 knockout mice. (Jin et al. 2000). 

This is suggestive that the absence of the FMRP protein causes misregulation of protein synthesis during synapse development and therefore produces the mental retardation phenotype associated with fragile X syndrome. (Jin et al. 2000). 

Much is still unknown about the precise mechanism that causes fragile X during the absence of FMRP. 

   

Figure 4 - The Functional Role of the FMRP Protein (extracted from Jin et al. Oxford Journals, 2000)

Figure 4 demonstrates an assumed model for FMRP function.

(A) In an unaffected individual, the FMRP dimerizes in the cytoplasm and enters the nucleus where it assembles into mRNP. mRNP interacts with specific RNA transcripts and other proteins. The mRNP particles consisting of FMRP and its target mRNA are transported to the cytoplasm and the mRNP is presented to the ribosome. Some of these FMRP-containing mRNPs in the neurons, are localised in the dendrites and mediate local protein synthesis.

(B) In fragile X patients without FMRP, the target mRNAs are able to interact with alternative mRNP. This permits partial translation but irregular regulation, localisation and abundance. 

 

Advances In Research on the Fragile X Syndrome:  

DNA testing has now replaced cytogenetic testing as the primary technique for the identification of the fragile X, however the efficacy associated with protein level screening has constantly been questioned. Past research have often found it difficult to address the ambiguous differentiation between premutation and full mutation genes. The phenotype of the full mutation has been well-established but is highly variable. (Mazzocco et al. 2000).

As of 2001 to present, research is still being conducted in an effort to identify the specific mRNA targets of FMRP and the effects that occur in the corresponding proteins when FMRP is absent. (Bardoni et al. 2001). 

Further studies based on the role of the FMR protein and its absence in fragile X is vital to the continued development of our understanding of fragile X syndrome and its molecular mechanism. (Mazzocco et al. 2000). 

Molecular genetic and neurobiological approaches are simultaneously required in order to develop effective intervention to fragile X syndrome. This involves producing new animal models for behaviour testing of the syndrome to provide further insight into the mechanisms of cognition, memory and behaviour in humans. This additional research can also potentially unlock secrets to the mechanisms involved in other disorders that also cause limiting cognition development. (Jin et al. 2000).

 

References:

 

* Jin, P and S. T. Warren (2000). "Understanding the Molecular Basis of Fragile X Syndrome". Human Molecular Genetics 9(6): 901-908.

 

* Mazzocco MM (2000). "Advances in Research on the fragile X syndrome". Mental Retardation and Developmental Disabilities Research Reviews 6(2): 96-106.

 

* Bardoni, B, A. Schenck, and J. L. Mandel (2001). "The Fragile X Mental Retardation Protein". Brain Research Bulletin 1;56(3-4): 375-82.

 

* Zalfa, F, M. Giorgi, B. Primerano, A. Moro, A. Di Penta, S. Reis, B. Oostra, and C. Bagni (2003). "The Fragile X Syndrome Protein FMRP Associates with BC1 RNA and Regulates the Translation of Specific mRNAs at Synapses" Cell, Vol. 112, 317-327.

 

* Pandey UB, S. R. Phadke, and B. Mittal (2004). "Molecular Diagnosis and Genetic Counseling for Fragile X Mental Retardation". Neurology India 52(1): 36-42.

 

* Barlow-Stewart, K (2007). "Fragile X Syndrome". The Australasian Genetics Resource Book (8th Ed.), published by the Centre for Genetics Education, 2007. Sourced from http://www.genetics.edu.au on 30/09/09.

 

* "Abnormal Development - Fragile X", UNSW Embryology (2009). Sourced from embryology.med.unsw.edu.au/Defect/fragilex.htm on 30/09/09. 

 

*"Fragile X Syndrome Blog" (2009). Sourced from blog.ahfr.org/2008/05/fragile-x-syndrome.html on 30/09/09.   

 

 

 

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