Fragile X syndrome and the future of psychiatry

1. Fragile X Syndrome
& The Future of Psychiatry
Dr Khalid Mansour
Mersey Care - 2019
Photo by Roger Ballen 1993: Dresie and Casie

2. Introduction

3. FXS: X-Linked LD
(Ensembl Genome Database Project; Lubs et al, 2012)
 LD: 2–3% of the population in the
industrialized world (Male-female>1.3-1)
 400 LD autosomal genes identified
(out of 19000).
 160 X-Linked LD disorders (out of
842):
◦ 81-syndromal, 50-non-syndromal;
◦ 102 genes identified,
 78-mapped (no genes identified),
 44 unmapped.
 X-Linked LD: 5–10% of males LD.
 FXS: 50% of X-linked LD.
Herbert Lubs

4. 81-SyndromalNon-syndromal
FXS: X-Linked LD
(Lubs et al, 2012)

5.  Online Mendelian Inheritance in
Man (OMIM) catalogue (March
2010) > 1640 references to LD
• X chromosome > 316 entities
• Similar sized chromosomes
• 6 > 50 references
• 7 > 58 references
• 8 > 60 references.
 Several authors > a relative
concentration of intelligence
genes on X chromosome
FXS: X-Linked LD
(Niranjan et al, 2015; Lubs et al, 2012)
Victor Mckusick
Johns Hopkins Hosp

6.  X-Linked LD: >> 5%–10% of LD in
males.
 FXS: 50% of X-linked LD
 FXS: most common cause of LD
2nd to Trisomy 21(Rousseau et al., 1995).
 Fragile X syndrome (FXS): most
described linked to LD and ASD.
 Multiple FXSs:
◦ FXS-A
◦ FXS-E
◦ FXS-F
FXS: X-Linked LD
(Lubs et al, 2012)

7. FXSs: (Lubs et al, 2012)
 >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
> on the 5’ untranslated region of the
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.

8. FXS: FMR1 gene
(Quartier, et al, 2017; Tabolacci, et al, 2016; Myrick et al, 2014; Vengoechea et al, 2012)

9. History

10. FXS: History: 1
(Lubs et al, 2012)
 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
Lionel Penrose
Julia Bell

11.  1983 Harrison et al > Xq27. 3 the precise
location of the fragile site.
 1990s: S Warren & Colleagues > FMRP
is a selective (suppressant) mRNA-
binding protein in dendrites.
 1991: Verkerk: FMR1 gene > FMRP
 1993: Ashley et al > Hyper-methylation >
silencing FMR1 gene
 1993: de Vries et al > A Prader-Willi-like
sub-phenotype of the FXS
 1994: Bakker et al > FMR1-KO Mice
model generated.
 1998: Murray et al > fragile X-associated
Premature Ovarian Failure.(also called
FXPOI)
Stephen Warren
Annemieke Verkerk
Anna Murray
FXS: History: 1
(Lubs et al, 2012)

12.  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: History: 1
(Lubs et al, 2012)

13. Epidemiology

14. Epidemiology:
(Hunter et al, 2014; a systematic review and meta-analysis)
 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

15.  FXS: The most common inherited LD.
 10% of undiagnosed male LD cases
 3% of undiagnosed female LD cases
 The most leading genetic cause of
autism.
 Second most common cause of LD
after Trisomy 21.
 FXS related mild dis. e.g. dyscalculia,
dyslexia, social phobia, and ADHD >
more common than FXS related LD
FXS: Other Statistics
(Rousseau et al., 1995; Hagerman et al, 2010; Paluszkiewicz et al, 2011)

16. Aetiology

17. FXS: Aetiology
(Quartier, et al, 2017; Myrick et al, 2014; Vengoechea et al, 2012)
Full Mutation FXS (200 or more CGG repeat expansions in
FMR1):
 In 99% of FXS : 200 CGG repeats expansion trigger total
Hyper-methylation of FMR1 > shut down > stop producing
FMRP > marked neurodevelopmental suppression
 In 1% of FXS : Partial or full Deletion / Point Mutation /
Micro-duplication > stop producing FMRP
Premutation FXS (55-199 CGG repeats expansion):
• Increased FMR1-mRNA (2–8 times) > toxic to cells.
• Reduced FMRP > partial neurodevelopmental suppression
◦ > Different cognitive and neuropsychological difficulties
◦ > Primary Ovarian Insufficiency in females couriers (40s-50s)
◦ > Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) in
male and female carriers (60s-70s)

18. FMR1 Gene & FMRP
(Grigsby, 2016; Levenga et al, 2010; Soden & Chen 2010)
 Fundamental importance for a
wide variety of mammalian
species (Oostra & Chiurazzi, 2001).
 Active early in foetal
development (Abitbol et al., 1993) in
brain, retina, liver, gonads, and
cartilage.
◦ RNA binding,
◦ mRNA shuttling,
◦ associating ribosomes with mRNA,
◦ DNA repair (Shi et al, 2012) >
◦ regulating normal neuronal
connectivity and plasticity (Lin 2015)
Lack of FMRP >
1. Downgraded
receptors
2. Suppression
of neuronal
transmission
3. Slow
transmission
in brain cells
4. Poor brain
development

19. Molecular Biomarkers in FXS
(Zafarullah & Tassone, 2019)

20. Full Mutation FXS: Interactions
(Rajaratnam et al, 2017; Iossifov et al, 2015)
Degrees of LD > FMRP produced;
CCG repeat number, mosaicism &
proportion of methylation.
Physical features: 80% > 1 or more
FXS, ASD & ADHD > Molecular
aetiologies intertwined,
◦ FXS > 20% of diagnosed ASD in
Monogenic disorders.
◦ Targeted treatments of FXS >
helpful for ASD (in animals).
◦ FMRP controls the translation of
approximately 30% of the genes
associated with ASD.

21. 1- Low level of FMRP > same but milder
forms of full-mutation FXS clinical
features
2- High level ab. FMR1mRNA >
Premature death of neurones:
 Toxic to sequestration of neuronal
proteins.
 Higher vulnerability to toxins e.g.
alcohol and pesticides.
 Intracellular calcium dysregulation
 Oxidative stress,
 Mitochondrial dysfunction
 Chronic DNA damage repair changes
 Formation of the toxic protein
FMRpolyG
3. High CGG repeats > Anticipation
Premutation-Associated Disorders
(Hagerman, 2018; Rajaratnam et al, 2017)

22. Number of
repeats
Allele
range
Phenotype Stability
<45 repeats
The most common
alleles contain 29
or 30 repeats
Normal (N) Normal
Transition to a full mutated allele has
never been reported. Extremely rare
cases of minor changes in repeat number
have been described
45–54 repeats
Intermediate
or grey-zone
allele (IA)
Normal
Possible instability upon transmission.
Very rare cases of expansion to a
premutation have been described.
Very rare cases of expansion to a full
mutation have been described in
two generations but not in one generation
55 to ∼200 repeats
without abnormal
methylation
Premutation
(P)
Risk of FXPOI for
females. Risk of
FXTAS for males
and females
Unstable upon transmission and at risk to
pass on a full mutation in one generation
when transmitted by a female. This risk is
proportional to the premutation size
>∼200 repeats
with abnormal
methylation
Full mutation
(M)
Males are affected
with FXS. ∼50% of
females are
affected with FXS
Biancalana et al, 2015

23. Inheritance

24. Mendelian Models
(Sherman et al, 2005)

25. 1-Epigenetics
(Zaidi et al, 2010)

26. (1-a) Epigenetics & Cell
Differentiation
(Suarez-Alvarez et al, 2012)

27. (1-b) Epigenetics & Environmental
Adaptations
(Norouzitallab et al, 2019)

28. (1-c) Epigenetics Mechanisms
(Triantaphyllopoulos et al, 2016; Alvarado et al, 2015)

29. (2) X-Inactivation / Dosage
Compensation
(Minks et al, 2008; Migeon, 1998)
heterochromatin
Mary Frances
Lyon

30. (3) Anticipation in FXS
(Grigsby, 2016: Hagerman 2013)
The greater the
number of
trinucleotide repeats,
the greater is the
probability of
expansion of the
gene to a
premutation or full
mutation allele.
These expansions
are most likely to
occur during female

31. (4) FXS: Mosaic Patterns
(Rajaratnam et al, 2017)
 Mosaic patterns > common in
males >
◦ Sperm mosaic: different sperms
have different sizes of the repeat
expansions.
◦ (Allele) Size mosaic:
 different sizes of the repeat
expansion in different cells.
 Most common form of mosaic
males.
◦ Methylation mosaic: Incomplete
methylation of a full mutation.

32. Clinical Features:
1- Full Mutation FXS

33. Clinical Features: Males (Ciaccio et al 2017: summery of 15 papers)
Long/Narrow face 83%
Macrocephaly 81%
Prominent ears 72–78%
High-arched palate 94%
Prominent jaw 80%
EEG anomalies 74%
Epilepsy 10–20%
Brain MRI anomalies Up to 50% of pts with
neuro. comorbidities
Psychomotor delay ~100%
ID ~100%
Aggressiveness 90%
Attention problems 74–84%
Hyperactivity 50–66%
Anxiety Disorder 58–86%
ASD 30–50%
Sleep problems 30%
ADHD 12–23%
Depression 8–12%
Joint hypermobility 50%
Pectus excavatum 50%
Flat feet 29–69%
Spine deformity 6–9%
Mitral valve anomalies 3–12%
Aortic root dilatation 25%
Refractive errors 17–59%
Strabismus 8–40%
Nystagmus 5–13%
Macroorchidism 63–95%
Obesity/overweight 53–61%
Recurrent otitis media 47–97%
Gastrointestinal complaints 31%

34. FXS: Common Clinical
Features
Michael Phelps

35. FXS: Common Clinical Features

36. Clinical Features:
2- Premutation FXS

37.  Children:
◦ Anxiety, ADHD, Social Deficits, mild cognitive & ASD
symptoms .
 Adults:
◦ 50% > anxiety and depression
◦ 35-40% (female) > premature ovarian insufficiency
(FXPOI).
◦ 40% (males) & 16% (females) > Fragile X-associated
Tremor/Ataxia Syndrome (FXTAS)
◦ Common > OCD, ADHD, Substance Abuse, Chronic
Fatigue & Chronic Pain.
◦ Also associated: Hypertension, Fibromyalgia,
Autoimmune Disorders & Sleep Problems.
Fragile X-Associated
Neuropsychiatric Disorders (FXAND)
(Hagerman, 2018; Rajaratnam et al, 2017; Iossifov et al, 2015)

38. (1) Fragile X-associated Tremor
Ataxia Syndrome (FXTAS)
(Hall et al, 2014)
 Urinary dysfunction
 Erectile dysfunction
 Constipation/faecal incontinence
 Orthostatic hypotension
 Depression, Anxiety
 Irritability, agitation, apathy
 Impaired olfaction
 Hearing loss
 Hypertension
 Sleep apnoea
 Neuropathy
 Muscle pain/fibromyalgia (in females)
 Hypothyroidism (in females)
Action tremor
Cerebellar Gait
Ataxia
Parkinsonism
Reflex Myoclonus
Executive
Dysfunction
Dementia

39. (2) Fragile X-associated
Primary Ovarian Insufficiency (FXPOI):
(Sullivan et al, 2005)
 20-25% of women (40 years or less) with
permutations (and 20% 40-45y old).
 Women with full mutation > same risk as
general public ∼1%.
 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.

40. FXS rare Phenotypes

41. Prader-Willi phenotype of FXS
(Muzar et al, 2018; Martínez‐Cerdeño et al, 2017)
 Several cases reported
of Prader-Willi
phenotype with genetic
abnormality of FXS and
no abnormality with
15q11–13 region.
◦ Most cases > FXS full
mutation
◦ Less cases > FXS
premutation (with FXTAS
too)

42.  47 XXX karyotype
 An expansion of
approximately 580
repeats in
the FRX gene on
two of her three X
chromosomes.
 Prader-Willi like
phenotype of FXS
Prader-Willi phenotype in Triple X with FXS
(Vandersteen et al, 2009)

43. Diagnosis
&
Genetic Tests

44. 1- Cytogenetic Testing:
 Conventional cytogenetic testing (Chromosome Analysis), (Karyotyping)
 Molecular Cytogenetics Testing via Fluorescence in-Situ Hybridization (FISH)
2- DNA/Genetic Tests: florescent/ radioactive probes
2-a- Specific (single) genetic text
 Southern Blot Analysis,
 Polymerase Chain Reaction (PCR):
◦ The Rapid Chain Reaction-Based Screening test
2-b- Arrays (high number group test):
 Microarray Comparative Genomic Hybridization (aCGH) Testing
 Single-nucleotide-polymorphism (SNP) genotyping array
 Next-generation Sequencing (NGS)
3- Immunocytochemical testing:
◦ The Methylation-Specific Melting Curve Analysis (MS-MCA):
◦ Willemsen Antibody Test.
Genetic Tests
(Vissers et al, 2016)

45. Cytogenetic Testing:
 Chromosome Analysis
(Karyotyping): a light
microscopy cytogenetic analysis
to examine metaphase or
prometaphase chromosomes
for structural anomalies.
 Fluorescence in-Situ
Hybridization (FISH): (largely
replaced by microarrays) > uses
labelled DNA probes to identify
sub-microscopic structural
chromosome anomalies—
microdeletions and
microduplications.
1- Conventional Cytogenetic Tests
(Vissers et al, 2016; Sharkey et al, 2005)

46. 2-a- DNA/Genetic Tests:
PCR (Polymerase Chain Reaction):
(Sherman et al, 2005)
Flanking primers > amplify DNA
fragments of repeat region >
approximate number of repeats
present in each allele.
Advantages:
 Accurate sizing of alleles in the
normal, “gray zone,” & small
premutation in a relatively short time.
 Assay not affected by X-chromosome
inactivation.
Disadvantages:
 Large mutations are more difficult to
amplify.
 No information about FMR1
methylation status

47. 2-a- DNA/Genetic Tests:
Southern Blot Analysis
(Sherman et al, 2005)
A methylation-sensitive
restriction enzyme > distinguish
between methylated &
unmethylated alleles.
Advantages:
 A crude measure of size of
repeat segments
An accurate assessment of
methylation status.
Disadvantages:
 More labour intensive than
PCR.
 Requires larger quantities of
genomic DNA.
 Inactivation of X-chromosome

48.  Array CGH compares DNA
from two differentially labelled
genomes; a test (or patient) vs
a reference (or control).
 Array CGH is similar to FISH
experiment but over hundreds
or thousands of loci and with a
much higher resolution.
 Depending on their design
(whole-genome arrays vs
targeted arrays), they have the
potential to detect the majority
of microscopic and sub-
microscopic chromosomal
2-b- DNA/Genetic Tests:
Microarray Comparative Genomic Hybridization (aCGH) Testing
(High number group genetic tests):
(Sherman et al, 2005; Karampetsou et al, 2014; Bejjani & Shaffer 2006)

49. 2-c- Immunocytochemical (ICC) Testing:
(Haenfler et al, 2018; Burry 2011)
 ICC is used to
anatomically visualize
localization of a protein
or antigen in cells by use
of a “primary
antibody” that binds to it.
The primary antibody
allows visualization of
the protein under
a fluorescence
microscope when it is
bound by a ”secondary
antibody” that has a

50.  Array tests: Molecular
Karyotyping, Multi-Gene Panel &
Exome Sequencing > in 337 ID
subjects vs standard clinical
evaluation.
 Standard clinical evaluation >
16% of cases (54/337) but only
70% of these (38/54) confirmed.
 Genomic > likely diagnosis in
58% (n=196) > exome
sequencing: 60%.
 Adoption of genomics as a first-
Routine Genetic Tests in LD (?)
( Anazi et al, 2017)

51. LD Genetic Tests
( Vissers et al, 2016)

52. Drug Treatments

53. Possible Drug Treatments (Berry-Kravis et al, 2018)

54. • Preclinical studies using
animal models of FXS have
yielded several agents that
rescue a wide variety of
phenotypes.
• Translation of treatments,
used in animal studies, to
humans with FXS has not
yet been successful,
shedding light a variety of
limitations with both animal
models and human trial
design.
Drug Trials: Conclusion
(Berry-Kravis et al, 2018; Erickson et al, 2018; Schaefer et al, 2015; Politte et al, 2013).

55. Preclinical studies in Fmr1-KO mice; outcome
(Berry-Kravis et al, 2018)

56. Clinical Trials since 2002 in FXS
(Berry-Kravis et al, 2018)
(Berry-Kravis et al, 2018)

57. Drug Development for FXS:
Lessons Learned: Oversimplistic Thinking
(Zafarullah & Tassone, 2019; Berry-Kravis et al, 2018)
Problems with
 Design,
 Assessment tools,
 Evaluating cognition,
 Evaluating disease
modification,
 Regulatory framework for
RCTs in children
 Preclinical safety
requirements
 Selection of clinical end
points

58. Molecular Biomarkers in FXS
(Zafarullah & Tassone, 2019)

59. Rescue of Fragile X Syndrome Neurons by DNA Methylation
Editing of the FMR1 Gene (Liu et al 2018)
 Recently developed DNA methylation editing tools to reverse hyper-
methylation event.
 Targeted demethylation of the CGG expansion by dcas9-
Tet1/single guide RNA (sgrna) switched the heterochromatin status
of the upstream FMR1 promoter to an active chromatin state,
restoring a persistent expression of FMR1 in FXS ipscs.
 Neurons derived from methylation-edited FXS ipscs rescued the
electrophysiological abnormalities and restored a wild-
type phenotype upon the mutant neurons.
 FMR1 expression in edited neurons was maintained in vivo after
engrafting into the mouse brain.
 Finally, demethylation of the CGG repeats in post-mitotic FXS
neurons also reactivated FMR1.
 Demethylation of the CGG expansion is sufficient
for FMR1 reactivation, suggesting potential therapeutic strategies for
FXS.

60. Reflections

61. FXS: a possible model for future psychiatry
RDoC studies (NIMH) (Insel et al, 2010).
ROAMER studies (Horizon 2020) (Schumann et al, 2014).
Future:
Less concepts like “Autistic Spectrum Disorders”
More concepts like “FXS Spectrum Disorders”

62. FXS Spectrum Disorders

63. Current thinking: [e.g. NIMH RDoC studioes (Insel et al, 2010),
ROAMER studies (Horizon 2020) (Schumann et al, 2014), CAN-MIND
(Rizvi et al, 2018): most psychiatrist disorders can
become as specific as FXS leading to more
objective and specific psychiatric management .
 Needs:
◦ Futuristic thinking and planning.
◦ Restructuring of psychiatry as a discipline
◦ Restructuring of training in psychiatry.
◦ Restructuring of services.
◦ More resources.
 Advances then will pay back very well.
FXS: a possible model for future
psychiatry

64. The Wheeler Family: TIME magazine: 2008
COMMENTS