1. 1. Fragile X Syndrome Some Recent Advances Dr Khalid Mansour Priory Cefn Carnau Roger Ballen 1993: Dresie and Casie
2. 2. Introduction: X-Linked LD
3. 3. FXS: X-Linked LD (Lubs et al, 2012) LD: 23% 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. 120 known fragile sites in the human genome > 6 sites on X chromosome > 3 linked to LD (Lukusa & Fryns, 2008). FXS-A = FSX = Martin-Bell syndrome: Band Xq27.3. > CGG repeats expansion > Gene (FMR1 & FMR4) > Protein (FMRP) > classical FXS FXS-E = FRAXE: (Gcz 2000) Band Xq27 > CCG repeats expansion > Gene (FMR2 & FMR3) (synonym AFF2) > Protein (???) > non-syndromic X-linked LD FXS-F = FRAXF: Band Xq28 > CGG repeats expansion > Gene (???) > Protein (???) > no clear phenotype has been established.
4. 5. FXS: History
5. 6. 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 X-linked LD (fragile x symptoms) although they did not know the cause > Martin-Bell Syndrome. 1953: Watson & Crick > DNA structure. 1969: Herbert Lubs: the first one to see the "marker X chromosome" in LD patients. 1970: Frederick Hecht: coined the term "fragile site > FXS. 1977: Grant Sutherland > Folate Deficient Medium 199 >specific FXS test JP Martin H Lubs
6. 7. FXS: History: 2 1991: Verkerk: FMR1 gene > FMRP 1991: Kerr et al > Nonspecific X Linked Mental Retardation (XLMR). 1993: Ashley et al > Hyper-methylation > silencing FMR1 gene 1990s: S Warren & Colleagues > FMRP is a selective (suppressant) mRNA-binding protein in dendrites. 1994: Bakker et al > FMR1-KO Mice model generated. 1998: Murray et al > fragile X- associated Premature Ovarian Failure.(also called FXPOI) S Warren
7. 8. 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. FXS: History: 3 Randi J. Hagerman
8. 9. FXS: Common Features
9. 10. 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
10. 11. FXS: Behavioural Symptoms LD (100%) (IQ: 35-70): with aging. 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
11. 12. FXS: Statistics
12. 13. 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 Syn.) (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) Fragile X Syndrome: Statistics Medscape reference 2013
13. 14. Statistics: USA Medscape reference 2013 Male FXS: 1 in 2500-4000. Male carriers: 1 in 250-800 Female FXS: 1 in 7000-8000. 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
14. 15. FXS: Aetiology
15. 16. FXS: Aetiology Medscape reference 2013 FXS Chromosomes > constriction of band Xq27.3. > site of Fragile X Mental Retardation-1 Gene (FMR1) FMR1 gene > produces Fragile X Mental Retardation Protein (FMRP) FMRP > a widely expressed mRNA-binding Translational Regulator with reportedly hundreds of potential targets.
16. 17. 1- DNA: Double stranded helix > Nucleotides > Nucleobase 2- RNA: 3-Proteins:
17. 18. FXS: FMR1 gene Medscape reference 2013 In FXS a full mutation in the FMR1 gene > Hyper-methylation of FMR1 gene > FMRP is not manufactured: (^Degree of methylation > ^degree of severity of FXS). Most commonly 29 - 30 repeats Premutation Full Mutation
18. 19. FMRP: regulatory protein of messenger RNA (mRNA) in neurons and dendrites Lack of FMRP > downgraded receptors in synapses. > suppression of neuronal transmission > slow transmission in the brain cells > poor brain development FXS: FMR1 gene Medscape reference 2013
19. 20. 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: Mutations & Premutation
20. 21. FXS: Mode of Inheritance
21. 22. FXS: Mode of Inheritance Medscape reference 2013 Females with full mutation: Unaffected mildly affected (LD, autism or physical features of FXS). (? X Inactivation) 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.
22. 23. 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
23. 24. Males with premutations: Individuals: Unaffected Mild FXS (LD, autism or physical features of FXS). 40%: FXTAS in old age. Daughters: exact premutation (no MOSAICS). FXPOI, FXTAS +/- mild FXS. Sons: unaffected (only get Y chromosome). FXS: Mode of Inheritance Medscape reference 2013
24. 25. - Most patients (98%) with FXS > CGG triplet expansion - Few patients (2%) > other abnormalities e.g. POINT MUTATION or DELETION of the FMR1 gene. FXS: Mode of Inheritance Medscape reference 2013
25. 26. 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.
26. 27. X Inactivation / Dosage Compensation
27. 28. FX- Associated syndromes
28. 29. 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,
29. 30. 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
30. 31. FXS: Genetic Tests
31. 32. 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/Genetic Tests: florescent/ radioactive probes 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. Genetic Tests Medscape reference 2013
32. 33. Genetic Tests Specific Chromosome Disorder: karyotyping / Chromosome analysis. Specific genetic disorder: Diagnosis: PCR or Southern Blot Analysis. FISH: labels the gene on the chromosome. Screening: Immunocytochemical testing e.g. Willemsen Antibody Test. The Rapid PCR-Based Screening test No specific genetic disorder: Microarray Comparative Genomic Hybridization (aCGH) Testing.
33. 34. FXS: Screening 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. The Rapid PCR-Based Screening test (Blood Spot Test ) (Tassone et al, 2008): Both males and females 55 to 200 CGG repeats & full-mutation ranges. Rapid detection using even 1% of the DNA from a single dried blood spot. Screening large populations. Costs $5 : $1 per sample. Flora Tassone
34. 35. ASURAGEN Amplide FMR1 PCR
35. 36. FXS: Drug Treatment
36. 37. FXS: Drug Treatment
37. 38. FXS: Drug Treatment
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40. 41. Plasticity: Long-Term Potentiation (LTP) & Long-Term Depression (LTD)
41. 42. In the normal state: mGluR activation by glutamate (glu) results in activation of dendritic translation through the phospholipase C (PL-C) cascade. FMRP levels increase with translational activation, and FMRP then inhibits translation, acting as the negative feedback or brake on the translational mechanism.
42. 43. When FMRP is missing in FXS: mGluR-mediated translation lacks the negative feedback balance > excessive: Synthesis of specific synaptic proteins, Internalization of AMPA receptors, Other synaptic changes > Excessive long-term depression. > persistently weak and immature synapses.
43. 44. MPEP & other mGluR5 ve modulator > blocks mGluR-mediated LTD Lithium: blocks inositol phosphate (IP) turnover, and blocking PL-C mediated signal transduction, also inhibits GSK3 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.
44. 45. Fenobam mGluR5 antagonist Open-label, single-dose trial in 12 adults. AFQ056 mGluR5 antagonist Phase II trials in adults and adolescents and Phase I trials in children underway Acamprosate Probable mGluR5 antagonist, Open-label study in 3 adult males. Phase III trial in children underway RO4917523 mGluR5 antagonist Phase II trials in adult males underway STX107 mGluR5 antagonist Phase II trials in adult males in development Riluzole believed to block presynaptic glutamate release. Open-label study in 6 adult males: Memantine NMDA antagonist Chart review of 6 young adults treated Minocycline MMP-9 inhibitor Phase II trials in children recently completed. Lithium GSK3 inhibitor; increases BDNF production Open-label study in 15 children and adults: Arbaclofen GABABR agonist Phase II randomized, placebo-controlled trial in 63 children and adults: Phase III trials in children and adults underway CX516 Positive modulation of AMPA receptors Randomized, placebo-controlled trials in 49 adults: OT Neuropeptide involved in pro- social behaviour Randomized, placebo-controlled trial in 10 young adult males: Donepezil Acetylcholinesterase antagonist Open-label trial in 9 adults. Randomized controlled trial in young adults underway
45. 46. In mice (& other animals) with 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). Drug Trials: Conclusion Politte et al, 2013
46. 47. In human trials: Not the same Results of targeted treatment trials have been encouraging but not striking. Treatment improves behaviours; do not reverse physical phenotype. Trials > increased methodological difficulties > significant potential for bias In animals: Both reduced and enhanced NMDAR functioning > ASD in mice. Why: Biological differences. More complex model: FXS > diverse symptoms, LD, ASD, ADHD (??). FXS Drug Trials: Conclusion Politte et al, 2013
47. 48. 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 Drug Trials: Conclusion Politte et al, 2013
48. 49. FXS: Management
49. 50. Critical Period
50. 51. Screening for FXS: Many FXS > late diagnoses > missing the critical period. 50% of parents > another child or pregnancy before diagnosis. Testing for FXS is recommended for FXS families and high risk groups: Features of FXS and LD. LD. Autism. Women with primary ovarian insufficiency. Aging adults with ataxia or tremor combined with other features of FXTAS. Family history consistent with FXS.
51. 52. New-born screening for FXS > currently researched in USA. Less-expensive screening methods have been developed > Blood Spot Test (Tassone et al, 2008): less than $5 / test > full mutation & premutation. If positive > further tests e.g. PCR or Southern Blotting. Screening for FXS:
52. 53. Risk for FXS > testing of the individual then family. Referral to a geneticist and/or genetic counselling: Identify individuals at risk Help with how information is conveyed to family members. review reproductive options for future pregnancies, including egg donation, prenatal diagnosis, adoption, and pre-implantation genetic diagnosis through polar body analysis. Help to be connected to support groups. Genetic Counselling (Hagerman et al, 2009)
53. 54. Educational Services (Hagerman et al, 2009) Research > educational services, have been associated with better behavioural outcomes and fewer autistic behaviours. Hagerman: I can make an FXS child able to learn but I can not teach him. MIND institute > New video games style educational programmes. Learning skills > information.
54. 55. Current research > IQ, symptoms and functioning of FXS patients improve with positive elements in: Environment. Stress management interventions. Sensory processing interventions. Behavioural intervention teams / programmes. Psychotherapy or Counselling. Speech therapy. Cognitive behavioural Therapy. Social Skills-Oriented Group Therapy. REHABILITATIVE INTERVENTIONS (Hagerman et al, 2009)
55. 56. The Wheeler Family: TIME magazine: 2008 COMMENTS