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1. 6. Discuss the genetics of Fragile X syndrome with special emphasis on the molecular basis of the disease.

   Trinucleotide Repeat Disorders Friday 4th September 2009 Stacey Sandell
2. Fragile X syndrome Fragile X syndrome is one of the most common forms of inherited mental retardation. 1 in 4000 males and 1 in 8000 females Characterised by mild to severe mental retardation (IQ between 20 and 60), mildly abnormal facial features (prominent jaw and large ears) and macroorchidism in post-pubescent males. >99% of mutations due to expansion of a CGG repeat in the 5’ untranslated region of the FMR1 gene FMR1 is located at Xq27.3 FRAXA (Fragile site, X chromosome, A site) and encodes the protein FMRP. X-linked dominant with reduced penetrance in females
3. Repeat size – normal range Repeat size 5-44 repeats Polymorphic but inherited in a stable fashion Trimodal distribution; 29 and 30 repeats are the most common. AGG interruption every 9/10 CGGs, these maintain the integrity of the repeat by preventing slippage.
4. Repeat size – intermediate range 45-58 repeats (Grey zone) Usually transmitted stably by females with occasional small increases or decreases in repeat size, especially in families with no family history of Fragile X A few studies have observed a higher degree of instability in paternal transmissions
5. Repeat size – premutation range 59-200 repeats Premutation alleles have no or only one AGG interruption Premutation alleles are unstable and can expand to a full mutation alleles through maternal transmission The likelihood for repeat instability increases with increasing CGG repeat number Premutation carriers were once considered as asymptomatic, however two distinct premutation associated phenotypes are now well documented Recent studies have reported higher rates of anxiety, hostility, psycoticism and obsessive-compulsive symptoms in premutation carriers
6. Premutation phenotype - females Higher incidence of Premature Ovarian Failure (POF) in female carriers POF defined as cessation of menstrual periods before age 40 This phenotype is specific to premutation carriers Studies have found that the risk of POF increases with increasing repeat size but only up to 100 repeats then the risk appears to plateau Premutation females need to be counselled for their risk of having an affected child and informed of their shortened reproductive life span
7. Premutation phenotype - males In 2001 a late-onset neurological disorder was described in five male premutation carriers Fragile X Tremor Ataxia Syndrome (FXTAS) Progressive intention tremor Cellebellar ataxia Cognitive decline Eosinophilicintranuclear inclusions in neurons throughout the brain in post-mortem samples Age-related penetrance reaching 47% in the age group 70-79yrs Less frequently and with a less severe phenotype described in female premutation carriers
8. Premutation – molecular mechanism of disease 1 In male premutation carriers increased levels of FMR1 mRNA are observed, but levels of FMRP are mildly reduced. There are many theories: Expansion leads to conformational changes that influence the initiation of translation Increased transcriptional activity due to feedback mechanism due to diminished translation More open promoter conformation and upregulation of transcription by CGG binding proteins (CGGBP1)
9. Premutation – molecular mechanism of disease 2 Histological studies of 4 symptomatic premutation carriers demonstrated: Neuronal degeneration in the cellebellum Eosinophilicintranuclear inclusions in neurons Inclusions showed a positive reaction with anti-ubiquitin antibodies Mouse knock-in model (98 repeats) showed: Elevated FMR1 mRNA levels Intranuclearinclusions with ubiquitin Increase in number and size of inclusions in specific brain regions over time.
10. Premutation – molecular mechanism of disease 3 A dominant RNA gain-of-function mechanism has been proposed as the molecular mechanism underlying FXTAS Elevated FMR1 transcripts might result in sequestration or misfolding of important cellular proteins with cumulative cytotoxic effect and formation of inclusions This theory may also apply to POF which could be considered a late-onset disorder, with a toxic effect on the follicles, further studies are required investigate this
11. Repeat size – Full mutation >200-several thousand repeats FMR1 becomes abnormally hypermethylated FMR1 silenced and no FMRP is produced Patients with deletions have confirmed that the syndrome is due to lack of FMR1 expression Mosaicism – may lead to higher functioning males Repeat size mosaicism Methylationmosaicism
12. Role of FMRP FMRP has two types of RNA-binding motif Two ribonucleoprotein K homology domains (KH domains) Clusters of arginine and glycine residues (RGG boxes) FMRP has nuclear localisation signal (NLS) and nuclear export signal (NES) FMRP has been found to associate with actively translating polyribosomes FMRP found in neuronal nucleoplasm and dendriticbranch points Abnormal dendritic spines were observed in both brains of Fragile X patients and FMR1 and knockout mice
13. Role of FMRP FMRP-associated mRNAs contain a sequence which forms a G-quartet structure Of 12 mRNA found to precipitate with FMRP and shown to be differentially expressed in Fragile X patients, 8 had a G quartet. Proposed model FMRP dimerises and enters the nucleus FMRP assembles into a complex with mRNP and interacts with specific RNA transcripts The complex is transported back out of the nucleus and associate with ribosomes to produce proteins Alternatively the complex is transported to the dendrites to regulate local protein synthesis in response to synaptic stimulation
14. Role of FMRP The mRNAs associated with FMRP that show differential expression do not all decrease in the absence of FMRP, some of them increase. Evidence for model 2 FMRP interacts with functional RNA-induced silencing complex (RISC) proteins FMRP interacts with Dicer, and work on drosophila has shown that lack of FMRP leads to a decrease in a neuron specific mature miRNA It is thought that FMRP facilitates the interaction between miRNAs and their target mRNA sequence, ensuring proper targeting of the miRNA-RISC and proper translational repression.
15. Role of FMRP