Fragile x What is New?

Fragile X SyndromeSome Recent AdvancesDr Khalid MansourPriory Cefn Carnau2013Roger Ballen 1993: Dresie and Casie

Introduction: X-Linked LD

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 FXSFXS-E = FRAXE: (Gcz 2000)Band Xq27 > CCG repeats expansion > Gene (FMR2 & FMR3) (synonym AFF2) > Protein (???) > non-syndromic X-linked LDFXS-F = FRAXF: Band Xq28 > CGG repeats expansion > Gene (???) > Protein (???) > no clear phenotype has been established.

FXS: History

FXS: History: 11938: Lionel Penrose first observed that more males than females in the population have LD (1.25:1) > X linked.1943: Martin andBell: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

FXS: History: 21991: Verkerk: FMR1 gene > FMRP1991: Kerret al> Nonspecific X Linked Mental Retardation (XLMR).1993: Ashley et al > Hyper-methylation > silencing FMR1 gene1990s: S Warren & Colleagues > FMRP is a selective (suppressant) mRNA-binding protein in dendrites.1994: Bakkeret al > FMR1-KO Mice model generated.1998: Murray et al > fragile X-associated Premature Ovarian Failure.(also called FXPOI)

S Warren

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

FXS: Common Features

FXS: Common Physical FeaturesElongated face & Broad foreheadLarge, prominent earsHigh arched palate Prominent jaw, Dental crowding Macro-orchidism (post-pubertal)Strabismus (squint)Murmur, Mitral valve prolapse, cardiomegaly, dilation of aortaHypotonia & joint laxity Flat feet, Hollow chest, Scoliosis

Michael Phelps

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 abnormalitiesMotor abnormalities

FXS: Statistics

FXS: The most common inherited LD (Paluszkiewicz et al, 2011).10% of undiagnosed male LD cases 3% of undiagnosed female LD casesThe 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: StatisticsMedscape reference 2013

Statistics: USA Medscape reference 2013Male FXS: 1 in 2500-4000. Male carriers: 1 in 250-800Female FXS: 1 in 7000-8000.Female carriers: 1 in 130-250Females 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)FMR1 gene > produces Fragile X Mental Retardation Protein (FMRP)FMRP > a widely expressed mRNA-binding Translational Regulator with reportedly hundreds of potential targets.

1- DNA: Double stranded helix >Nucleotides > Nucleobase 2- RNA:3-Proteins:

FXS: FMR1 gene Medscape reference 2013 In FXS a full mutation in theFMR1gene > Hyper-methylation ofFMR1 gene > FMRP is not manufactured: (^Degree of methylation > ^degree of severity of FXS).

Most commonly 29 - 30 repeatsPremutationFull Mutation

FMRP: regulatory protein of messenger RNA (mRNA) in neurons and dendritesLack 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

55-199 repeats: PREMUTATION > enhanced production ofFMR1-mRNA (28 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

FXS: Mode of Inheritance

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: FXS50% girls: carriers with full mutation. Females with premutations: 20-25%: Primary Ovarian Insufficiency. 4-8%: FXTASIncreased risk of autoimmune disorders (hypothyroidism & fibromyalgia).CGG triplets are UNSTABLE > boys and girls > full mutation or premutations.

Males with a full mutation: Individuals: have full FXS. Sons: are unaffected > only get Y chromosomeDaughters: 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: UnaffectedMild 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

- Most patients (98%) with FXS > CGG triplet expansion - Few patients (2%) > other abnormalities e.g. POINT MUTATION or DELETION of the FMR1gene. FXS: Mode of Inheritance Medscape reference 2013

FXS: Mosaic PatternsMosaic 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.

X Inactivation / Dosage Compensation

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 inclusionsPeripheral 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 failureEarly menopause Irregular menses, Decreased fertility, Elevated FSH

FXS: Genetic Tests

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 probesPolymerase chain reaction (PCR):The Rapid Polymerase Chain Reaction-Based Screening testSouthern Blot Analysis, Immunocytochemical testing:The methylation-specific melting curve analysis (MS-MCA):Willemsen Antibody Test.Genetic Tests Medscape reference 2013

Genetic TestsSpecific 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 testNo specific genetic disorder: Microarray Comparative Genomic Hybridization (aCGH) Testing.

FXS: Screening TestsSabaratnam & 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

ASURAGEN AmplideFMR1PCR

FXS: Drug Treatment

FXS: Drug Treatment

FXS: Drug Treatment

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Plasticity: Long-Term Potentiation (LTP) & Long-Term Depression (LTD)

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.

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.

MPEP & other mGluR5 ve modulator > blocks mGluR-mediated LTDLithium: 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.

Table 1.Comparison of Phase II and III clinical trials in FXS.CompoundMechanism of actionClinical findingsFenobammGluR5 antagonistOpen-label, single-dose trial in 12 adults.AFQ056mGluR5 antagonistPhase II trials in adults and adolescents and Phase I trials in children underwayAcamprosateProbable mGluR5 antagonist, Open-label study in 3 adult males.Phase III trial in children underwayRO4917523mGluR5 antagonistPhase II trials in adult males underwaySTX107mGluR5 antagonistPhase II trials in adult males in developmentRiluzolebelieved to block presynaptic glutamate release.Open-label study in 6 adult males: MemantineNMDA antagonistChart review of 6 young adults treated MinocyclineMMP-9 inhibitorPhase II trials in children recently completed.LithiumGSK3 inhibitor; increases BDNF productionOpen-label study in 15 children and adults: ArbaclofenGABABR agonistPhase II randomized, placebo-controlled trial in 63 children and adults: Phase III trials in children and adults underwayCX516Positive modulation of AMPA receptorsRandomized, placebo-controlled trials in 49 adults: OTNeuropeptide involved in pro-social behaviourRandomized, placebo-controlled trial in 10 young adult males: DonepezilAcetylcholinesterase antagonistOpen-label trial in 9 adults. Randomized controlled trial in young adults underway

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: ConclusionPolitte et al, 2013

In human trials: Not the sameResults 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 biasIn 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

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 repeatsExtent of methylationAssociated pathology Stage of development Still many other potential therapies are to be discovered Drug Trials: ConclusionPolitte et al, 2013

FXS: Management

Critical Period

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.

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:

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)

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.

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)

The Wheeler Family: TIME magazine: 2008COMMENTS