A Diagnostic Approach!!

1. FRAGILE X
SYNDROME
DIVYA NARAYANAN
OCT 13TH, 2014

2. HISTORY
1943 - Discovery
by Martin & Bell
1969 - Development
of Chromosome Tests
by Herbert Lubs
1999 – FMR1 gene
and mutational basis
discovered

4. FMR1 5’ LEADER

5. FMR1 GENE

6. METHYLATION OF CYTOSINE LEADS TO
TRANSCRIPTIONAL SILENCING IN FXS

7. CARRIERS ARE MORE PREMUTATION AFFECTED

8. CHARACTERIZATION OF
PREMUTATION CGG EXPANSION

9. WHEN THE CGG EXPANSION HAPPENS?

10. TYPES OF FMR1 REPEAT EXPANSION MUTATIONS
Type No. of CGG
repeats
Methylation
status of FMR1
Phenotype
in males
Phenotype in
females
Normal 5-50 Unmethylated Unaffected Unaffected
Premutation 51-200 Unmethylated Unaffected Unaffected
Full mutation >200 Hypermethylated Affected 50% affected;
50% unaffected
Mosaicism Varies 51 to
>200
Partially
methylated
Affected Highly variable
Methylation
Mosaicism
>200 Partially
methylated
Affected Highly variable
Unmethylated
full mutation
>200 Unmethylated Nearly all
affected
Highly variable

11. FMRP PRESENCE

12. FMRP ABSENCE

13. FRAGILE X SYNDROME INDIVIDUALS EXHIBIT
ALTERED DENDRITIC SPINE MORPHOLOGY
Weiler et al. Am J Med Genet (1999)

14. DIAGNOSIS
Pedigree analysis

15. DIAGNOSIS
Karyotyping

16. DIAGNOSIS
Direct DNA analysis

17. DIAGNOSIS
Southern Blotting

18. DIAGNOSIS
Immunocytochemical tests

19. Sample : Maternal Blood;
amniotic fluid
Tests : PCR, Southern Blot;
Karyotype, FISH
analysis
Enzymes : EcoRI; EagI
Results : a) Deletion in one FMR1
gene in Linda
b) one 33 CGG repeat
allele (normal) and one
110 CGG repeat allele
(premutation) in fetus
CASE STUDY
35 years old
10 years old (first child)

20. CASE STUDY
1. Molecular size marker
2. Control (female with premutation)
3. Control (female with full mutation)
4. Normal male
5. Linda’s result
6. Linda’s fetus result
7. Female with intermediate result
8. Female with normal result
9. Male with full mutation

21. CASE STUDY
Electropherogram
Largest peak : 33 CGG repeats
Smallest peak : 110 CGG repeats

22. CASE STUDY
• After PCR analysis, how many repeat
length results are expected?
• What steps can be taken by the
laboratory to investigate this apparent
discrepancy?
• Is Linda a carrier of fragile X syndrome?
• Is amniotic fluid an appropriate specimen
type for prenatal fragile X testing?
• Should these results alleviate Linda’s
parental anxiety?

23. REFERENCES
• Weiler et al, Synaptic Synthesis of the Fragile X Protein: Possible Involvement in Synapse Maturation
and Elimination; American Journal of Medical Genetics 83:248–252 (1999)
• Dobson et al, Identifying intrinsic and extrinsic determinants that regulate internal initiation of
translation mediated by the FMR1 5' leader; BMC Molecular Biology 2008, 9:89 doi:10.1186/1471-
2199-9-89
• Tassone et al, Elevated Levels of FMR1 mRNA in Carrier Males: A New Mechanism of Involvement in
the Fragile-X Syndrome; Am. J. Hum. Genet. 66:6–15, 2000
• Haas, History Tends to Repeat: FMR-1 Silencing in Fragile X Syndrome; Eukaryon, Vol. 3, February
2007
• Berman et al, Mouse Models of the Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS) and the
Fragile X Premutation; Movement Disorders: Genetics and Models, second edition
• Jin et al, RNA-Mediated Neurodegeneration Caused by the Fragile X Premutation rCGG Repeats in
Drosophila; Neuron, Vol. 39, 739–747, August 28, 2003
• Cheng et al, Astrocytes and Developmental Plasticity in Fragile X; Neural Plasticity Volume 2012,
Article ID 197491,12 pages doi:10.1155/2012/197491
• Fragile X syndrome, Atlas of Genetic Diagnosis and Counselling (2006)
• Eliez et al, Genetics of Childhood Disorders: XI. Fragile X Syndrome; J . Am. Acad. Child Adolesc.
Psychiatry, 39:2. February 2000
• Jaquemont et al, Fragile X Premutation Tremor/Ataxia Syndrome : Molecular, clinical and
Neuroimaginf Correlates; Am J Hum Genet. Apr 2003; 72(4): 869-878
• Damell et al, FMRP Stalls Ribosomal Translocation on mRNAs Linked to Synaptic Function and
Autism; Cell 146, 247–261, July 22, 2011
• Heim, Fragil X syndrome; Diagnostic Molecular Pathology in Practice, 2011

24. THANKYOU