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Fragile X Syndrome Some Recent Advances. Dr Khalid Mansour Castlebeck: NE . Roger Ballen 1993: Dresie and Casie. Introduction: X-Linked LD. FXS: X-Linked LD ( Lubs et al, 2012 ). LD: 2–3% of the population in the industrialized world ( Male to female ratio: 1.3 : 1)
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Fragile X SyndromeSome Recent Advances Dr Khalid Mansour Castlebeck: NE Roger Ballen 1993: Dresieand Casie
Introduction: X-Linked LD
FXS: X-Linked LD(Lubset al, 2012) • LD: 2–3% of the population in the industrialized world (Male to female ratio: 1.3 : 1) • X-Linked LD: 5%–10% of LD in males. • FXS: 50% of X-linked LD. (Rousseau et al., 1995). • 160 X-Linked LD disorders: 102 genes, 81 syndromal & 35 non-syndromal; 78 mapped. • <400 LD autosomal genes have been identified. • Fragile X syndrome (FXS): most described. • Multiple FXSs: FXS-A, FXS-E & FXS-F
FXSs: • FXS-A = FSX = Martin-Bell syndrome: • Band Xq27.3. > CGG repeats expansion > Gene (FMR1 & FMR4) > Protein (FMRP) > classical FXS • FXS-E = FRAXE: (Gécz 2000) • Band Xq27 > CCG repeats expansion> Gene (FMR2 &FMR3) (synonym AFF2) > Protein (???) > non-syndromicX-linked LD • FXS-F = FRAXF: • Band Xq28 > CGG repeats expansion > Gene (???) > Protein (???) > no clear phenotype has been established.
FXS: History
FXS: History: 1 • 1938: Lionel Penrose first observed that more males than females in the population have LD (1.25:1) > X linked. • 1943: Martin and Bell: described a described a family with 11 members with fragile x symptoms although they did not know the cause > recognition of Martin-Bell Syndrome. • 1953: Watson & Crick > DNA structure. • 1969: Herbert Lubs: • Sighted an unusual "marker X chromosome" in association with LD • Developed the chromosomal test for it. JP Martin H Lubs
FXS: History: 2 • 1970: Frederick Hecht: coined the term "fragile site“ > FXS. • 1991: Verkerk:FMR1 gene > FMRP • 1991:Kerr et al > “Non‐specific X Linked Mental Retardation” (XLMR). • 1993: Ashley et al > Hyper-methylation > silencing FMR1 gene • 1994:Bakker et al > FMR1-KO Mice model generated. • 1998:Murray et al > fragile X-associated Premature Ovarian Failure.(also called FXPOI)
FXS: History: 3 • 2001: Hagerman et al > Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) • 2002:Huber et al > mGluR-LTD exaggerated in FMR1-KO Mice • 2004:Bear et al > mGluR theory of FXS. • 2005: Yan et al > MPEP improves FXS in animals. • 2009: Clinical trials in humans. Randi J. Hagerman
FXS: Common Features
FXS: Common Physical Features • Elongated face & Broad forehead • Large, prominent ears • High arched palate • Prominent jaw, Dental crowding • Macro-orchidism (post-pubertal) • Strabismus (squint) • Murmur, Mitral valve prolapse, cardiomegaly, dilation of aorta • Hypotonia & joint laxity • Flat feet, Hollow chest, Scoliosis Michael Phelps
FXS: Behavioural Symptoms • LD (IQ: 35-70): with age. • ASD (50 – 60%) • ADHD (30 – 60%) • Epilepsy (5 – 20%) (mostly TLE) • DSH (10 – 30%) • Aggression(15 – 35%) • Prader-Willi phenotype. • Sensory processing disorder. • Psychosis • Speech abnormalities • Motor abnormalities
FXS: Statistics
Fragile X Syndrome: StatisticsMedscape reference 2013 • FXS: The most common inherited LD (Paluszkiewicz et al, 2011). • 10% of undiagnosed male LD cases • 3% of undiagnosed female LD cases • The most leading genetic cause of autism (Paluszkiewicz et al, 2011). • Second most common cause of LD after Trisomy 21 (Down S.) (Rousseau et al., 1995). • FXS related milder problems (e.g. dyscalculia, dyslexia, social phobia, and ADHD) may be more common than FXS related LD (Hagerman et al, 2010)
Statistics: USA Medscape reference 2013 • Male FXS: 1 in 2500-4000. • Female FXS: 1 in 7000-8000. • Male carriers: 1 in 250-800 • Female carriers: 1 in 130-250 • Females with FXS: less LD and less physical characteristics. • Males with FXS: more likely to be sensitive to environmental factors. • Mortality rate: not affected
FXS: Aetiology
FXS: AetiologyMedscape reference 2013 • FXS Chromosomes > constriction of band Xq27.3. • > site of “Fragile X Mental Retardation-1 Gene” (FMR1) • FMR1gene > produces “Fragile X Mental Retardation Protein (FMRP) • FMRP > a widely expressed mRNA-binding Translational Regulator with reportedly hundreds of potential targets.
FXS: FMR1 geneMedscape reference 2013 • In FXS a full mutation in the FMR1 gene > • Hyper-methylation of FMR1gene > • FMRP is not manufactured: (Degree of methylation > degree of severity of FXS).
FXS: FMR1 geneMedscape reference 2013 • FMRP: regulatory protein of messenger RNA (mRNA) in neurons and dendrites • Lack of FMRP >downgradedreceptors in synapses. • > suppression of neuronal transmission • > slow transmission in the brain cells • > poor brain development
FXS: Mutations & Premutation • 55-199 repeats: PREMUTATION > enhanced production of FMR1-mRNA (2–8 times normal levels). • > Primary Ovarian Insufficiency (40s-50s) • > Fragile X-associated Tremor/Ataxia Syndrome(FXTAS). (60s-70s) • 200 repeats or more: FULL MUTATION > • Hyper methylation of the repeats in the FMR1 region > • Reduced or absent FMR1-mRNA > • Decreased or absent Fragile X Mental Retardation Protein (FMRP) > FXS.
FXS: Mode of Inheritance
FXS: Mode of InheritanceMedscape reference 2013 Although most patients with FXS have a CGG triplet expansion > few patients have a POINT MUTATION in the FMR1 gene or a DELETION of the gene.
FXS: Mosaic Patterns • Mosaic patterns > common in males > unstable number of repeats over generations > pattern of inheritance difficult to predict. • (Allele) Size mosaic: • different sizes of the repeat expansion in different cells. • Most common form of mosaic males. • Sperm mosaic: different sperms have different sizes of the repeat expansions. • Methylation mosaic: Incomplete methylation of a full mutation.
FXS: Mode of Inheritance Medscape reference 2013 Females with full mutation: • Unaffected – mildly affected (LD, autism or physical). • 50% boys: FXS • 50% girls: carriers with full mutation. Females with premutations: • 20-25%: Primary Ovarian Insufficiency. • 4-8%: FXTAS • Increased risk of autoimmune disorders (hypothyroidism & fibromyalgia). • CGG triplets are UNSTABLE > boys and girls > full mutation or premutations.
FXS: Mode of Inheritance Medscape reference 2013 Males with a full mutation: • Individuals: have full FXS. • Sons: are unaffected > only get Y chromosome • Daughters: mutations or premutation to one X chromosome (sperm: MOSAICS). • Most closely resembles X-linked dominance with partial penetrance (see Dobyns et al 2004).
FXS: Mode of Inheritance Medscape reference 2013 Males with premutations: • Individuals: • Unaffected • Mild FXS (LD, autism or physical). • 40%: FXTAS in old age. • Daughters: • exact premutation (no MOSAICS). • FXPOI, FXTAS +/- mild FXS. • Sons: unaffected (only get Y chromosome).
FX- Associated syndromes
Fragile X-associated tremor ataxia syndrome (FXTAS): • 33-46% of men, with permutations, older than 50 years. • 4-8% in older women with permutations . • Other signs of neurodegeneration: • Brain atrophy, • Middle cerebellar peduncle lesions. • Intranuclear inclusions • Peripheral neuropathy, • Autonomic dysfunction • Clinical features of FXTAS include: • Cerebellar ataxia, • Dementia, • Anxiety, Irritability, Depression, • Incontinence, • Impotence,
Fragile X-associated Primary Ovarian Insufficiency: • Reported in 20-25% of women with permutations; • 30-fold increase compared with the general population. • Women with a diagnosis of ovarian insufficiency: 2-15% have a permutation of FXS. • Directly related to the number of CGG repeats: • Premature ovarian failure • Early menopause • Irregular menses, • Decreased fertility, • Elevated FSH
FXS: Genetic Tests
FXS: Genetic Tests Medscape reference 2013 Cytogenetic Testing: • Conventional cytogenetic testing or (chromosome analysis), (karyotyping): • Molecular Cytogenetics Testing via Fluorescence in-Situ Hybridization (FISH) • Microarray Comparative Genomic Hybridization (aCGH) Testing DNA/GeneticTests: • Polymerase chain reaction (PCR): • The Rapid Polymerase Chain Reaction-Based Screening test • Southern blot analysis, • Immunocytochemical testing: • The methylation-specific melting curve analysis (MS-MCA): • Willemsen "antibody test“:
FXS: Genetic Tests Sabaratnam & Thakker, 2003; Medscape reference 2013 Polymerase Chain Reaction (PCR): • Is the routine screening test used on FXS. • Faster, less expensive & requires a minimal sample, • Effective for small premutations but not very sensitive in full mutations. • Recent success with fluorescent methylation-specific PCR and Gene Scan Analysis may further expand diagnostic options. Southern Blot Analysis: • Can detect full mutations, methylation status & presence of mosaicism. • It is more labour-intensive than PCR and requires larger amounts of genomic DNA. • Southern blot analysis detects alleles in all size ranges, but precise sizing is not possible.
FXS: Genetic Tests Sabaratnam & Thakker, 2003; Medscape reference 2013 Immunocytochemicaltesting: • Can be used in males with full mutations only • Allow rapid and reliable identification of patients • Not useful in premutations, mosaic males or females. The methylation-specific melting curve analysis (MS-MCA): • relies on the fact that the transcription errors seen on the FMR1 gene results in hypermethylation of the genetic material (Dahl et al., 2007). Willemsen "antibody test“: (Willemsen et al, 1995) • a diagnostic test based on use of antibody to FMR protein (product of FMR-1 gene) to detect presence or absence of FMR protein in lymphocytes or hair root.
The Rapid Polymerase Chain Reaction-Based Screening test (Tassone et al, 2008): • is applicable for screening both males and females • is applicable for allele sizes throughout the premutation (55 to 200 CGG repeats) and full-mutation ranges. • is capable of rapid detection of expanded alleles using as little as 1% of the DNA from a single dried blood spot. • is suitable for screening large populations of new-born or those at high risk. • The test described herein costs less than $5 per sample for materials; with suitable scale-up and automation, the cost should approach $1 per sample. Flora Tassone
FXS: Drug Treatment
Plasticity: Long-Term Potentiation (LTP) & Long-Term Depression (LTD)
In the normal state: • mGluRactivation by glutamate (glu) results in activation of dendritic translation through the phospholipase C (PLC) cascade. • FMRP levels increase with translational activation, and FMRP then inhibits translation, acting as the negative feedback or “brake” on the translational mechanism.
When FMRP is missing in FXS: • mGluR-mediated translation lacks the negative feedback balance >excessive: • synthesis of specific synaptic proteins, • internalization of AMPA receptors, and • other synaptic changes • > excessive long-term depression and • > persistently weak and immature synapses.
MPEP & other mGluR5 –ve modulator > blocks mGluR-mediated LTD • Lithium: blocks inositol phosphate (IP) turnover, and blocking PL-C mediated signal transduction, also inhibits GSK3β activity > block in part excessively activated mGluR-mediated translation. • CX516 or other Ampakines: increases AMPA activity directly & redistributes AMPA receptors to the synaptic membrane through activation of BDNF.
Drug Trials: Conclusion Politte et al, 2013 • In animal models of FXS, data supporting the mGluR theory and drugs that correct mGluR overexpression are robust, with many studies reporting phenotypic ‘rescue' and behaviour that is indiscernible from WT.
FXS Drug Trials: Conclusion Politteet al, 2013 • In human trials: not the same • Results of targeted treatment trials have been encouraging but less striking. • Both reduced and enhanced NMDAR functioning > ASD in mice. • Treatment improves behaviours; do not reverse physical phenotype. • Trials > increased methodological difficulties > significant potential for bias • FXS is associated with a diverse symptoms, LD, ASD, ADHD (??).
Drug Trials: Conclusion Politte et al, 2013 • Most favourable outcomes would be obtained with early childhood intervention, • The choice of target population in future clinical trials should be carefully considered. • Number of CGG repeats • Extent of methylation • Associated pathology • Stage of development • Still many other potential therapies are to be discovered
FXS: Management