Fragile X Syndrome is caused by mutations in the FMR1 gene located on the X chromosome. The most common mutation is a CGG triplet expansion in the 5' untranslated region of the FMR1 gene, which causes hypermethylation and silencing of the gene. This results in reduced or absent production of the fragile X mental retardation protein (FMRP) and leads to excessive mGluR signaling in synapses. Several drug treatments aim to reduce mGluR activity and correct the imbalance, such as mGluR antagonists and drugs that increase GABA or AMPA receptor activity. While data from animal studies of Fragile X Syndrome support the mGluR theory and effectiveness of related drug treatments

1. Fragile X Syndrome
Some Recent Advances
Dr Khalid Mansour
Priory Cefn Carnau
Roger Ballen 1993: Dresie and Casie

2. Introduction:
X-Linked LD

3. 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)
 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

4. FXSs:
 >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: (Gécz 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.

5. FXS:
History

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

7. FXS: History: 2
 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
 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

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

9. FXS:
Common Features

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

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

12. FXS:
Statistics

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

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

15. FXS:
Aetiology

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.

17. 1- DNA: Double stranded helix >
Nucleotides > Nucleobase
2- RNA:
3-Proteins:

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

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

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

21. FXS:
Mode of Inheritance

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.

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

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

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

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.

27. X Inactivation / Dosage Compensation

28. FX-
Associated syndromes

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,

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

31. FXS:
Genetic Tests

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

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.

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

35. ASURAGEN Amplide FMR1 PCR

36. FXS:
Drug Treatment

37. FXS: Drug Treatment

38. FXS: Drug Treatment

39. 39

40. 40

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

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.

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.

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 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.

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

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

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

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

49. FXS:
Management

50. Critical Period

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.

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:

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)

56. 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.

57. 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)

58. The Wheeler Family: TIME magazine: 2008
COMMENTS