Louise Stanley

1. Fragile X is a clinically a highly heterogeneous disorder. What is the genetic basis of this heterogeneity? Describe a practical testing strategy for a diagnostic laboratory. Louise Stanley

2. Key Words • FMR1 – Fragile X Syndrome, FXTAS, POF • CGG expansion in 5’UTR • Disease mechanisms – loss of function, RNA gain-of-function • Testing by fluorescent PCR and Southern Blotting

3. FMR1 Disorders • Caused by CGG expansion in 5’UTR of FMR1 FMR1 [Xq27.3] 3 Disorders Fragile X Syndrome POF FXTAS Increasing Age for disease phenotype onset

4. Fragile Sites on X Chromosome • Can’t distinguish between FRAXA (FMR1), FRAXE (FMR2), FRAXF or FRAXD fragile sites • [FRAXE - GCC repeat in 5’ untranslated region (UTR) of the FMR2 gene. Transcriptional silencing due to methylation of promoter region – clinical phenotype milder than FRAXA – few diagnostic labs investigate]

5. Allele size ranges – FMR1 >200 ~51 - 58 ~59 - 200 5 - ~ 50 Intermediate Premutation Normal Full FXTAS/POF FXS No associated phenotype • Normal and intermediate length FMR1 alleles are more prone to expand into premutations during PATERNAL transmission • Expansion of premutations into full mutation occurs almost exclusively during MATERNAL transmission • Males only pass on premutations in their sperm – inherit full mutation from their mother harbour expanded allele in somatic cells but do not transmit expanded allele to their progeny – shortening of large CGG tracts in spermatogonia occurs around 13 to 17 weeks of fetal development – dependant on replication based mechanism

6. Fragile X Syndrome (FXS) • X-linked disorder caused by CGG expansion in the 5’UTR of FMR1 • Common cause of mental retardation in both males and females (males generally more severely affected than females) • ~1 in 4000 males, ~ 1 in 8000 females • Clinical features • Moderate to Severe intellectual and social impairment • Large ears and head • Long face • Macroorchidism

7. Fragile X Syndrome (FXS) • FXS - >200 repeats in FMR1and methylation of promoter region occurs - transcriptional silencing and loss of function of FMRP [minority of cases ~ 1% of FXS caused by point mutations or small deletions in FMR1] • Immunocytochemical tests can detect presence/absence of FMRP in lymphocytes – males with methylated full mutation no FMRP produced – can’t differentiate between normal and pre-mutation alleles • FMRP (Fragile X Mental Retardation Protein) – normal role is as a suppressor of target mRNA translation via binding of non-coding RNA structures • FMRP may regulate translation of its mRNA through miRNA interaction • Skewed X-inactivation can be a modifier of the phenotype observed in female patients

8. Fragile X Syndrome (FXS) • Sherman’s Paradox Normal Transmitting Male ~100 rpts All female offspring obligate carriers Carrier Female ~100 rpts Probability of expansion during transmission to offspring from females carriers is almost certain if premutation is 90 repeats or longer Affected male offspring >200 repeats • Most FMR1 alleles the sequence of CGG trinucleotide repeats is interrupted by an AGG repeat at repeat 9 or 10 and 19 or 20 (and occasionally repeat 30). • These AGG repeats appear to "anchor" the segment against repeat expansion, probably by disruption of DNA secondary structures that may form during DNA replication. • Sequences of uninterrupted CGG repeats beyond the last AGG repeat ("pure CGG trinucleotide repeats") greater than approximately 33-39 triplets appear to increase the instability of maternal alleles and increase the risk for expansion of the number of trinucleotide repeats on transmission to offspring

9. FXTAS/POF • Alleles in the premutation range (~59-200)are not associated with FXS (may be some evidence that premutations can cause very mild FXS-like symptoms but debatable) but can be associated with Fragile X Associated Tremor/Ataxia Syndrome (FXTAS) and premature ovarian failure (POF) • FXTAS affects greater number of males than females • POF females only • Disease pathogenesis for FXTAS by RNA gain-of-function • Disease mechanism for POF uncertain

10. FXTAS • Clinical features include: • Cerebellar degeneration and loss of Purkinje cells • Primary neuropathological hallmark is easinophilic ubiquitin positive intranuclear inclusions disseminated through brain and spinal column • Progressive cognitive decline ranging from executive and memory deficits to dementia • Some cases have also presented as a multi-system neurodegenerative disorder • Age in Years Risk (FXTAS in males) • 50-59 17% • 60-69 38% • 70-79 47% • ≥80 75%

11. FXTAS – Disease Mechanism • FMR1 mRNA levels increased 2-10 fold in FXTAS – FMRP levels are normal/slightly lower • FXTAS largely restricted to carriers of pre-mutations – suggests that FMR1 gene must be transcriptionally active for disease • Severity of both clinical and neuropathological phenotypes correlate with extent of CGG-repeat expansion size

12. FXTAS – Disease Mechanism • Evidence to support RNA gain-of-function in FXTAS includes • Increased levels of CGG containing FMR1 mRNA – ribonuclear inclusion formation • “Knock-in” mouse model with ~100 CGG repeats results in intranuclear inclusions present in brain, increase in both size and number over life course • Significant association between length of CGG tract and frequency of intranuclear inclusions • Intranuclear inclusions formed in primary neural progenitor cells and established neural cell lines using reporter constructs with premutations • Proposed that rCGG repeat binding proteins become sequestered to lengthy rCGG repeats – mechanistically similar to DM1 • Identified binding proteins include: • Pura • hnRNP A2/B1 • CUGBP1 • Affect transcription, mRNA trafficking, splicing and translation Direct interaction Indirect through interaction with hnRNP A2/B1

13. FXTAS – Disease Mechanism • Possible involvement of antisense transcripts • ASFMR1 encodes ncRNAs that are transcribed from two alternative promoters • One promoter is bidirectional and produces a transcript, also named FMR4, which may possess anti-apoptotic function • The other drives the major transcription initiation site in premutation cells, is located in FMR1 intron 2, and produces transcript that overlaps the CGG repeat region • Function of these antisense transcript remains unclear

14. Testing Strategy Sample Arrives (M + F) Patients with confirmed FH get analysed by PCR and always get Southern Blot analysis to exclude presence of mosaicism Blood stored in case manual extraction required Automated DNA Extraction (unless confirmed FH – probe) Fluorescent PCR to size CGG rpt in FMR1 Females Males 1 normal sized allele Premutation No allele detected Homozygous normal allele/N + premutation 2 normal sized alleles Repeat PCR Southern Blot Report Report Southern Blot Failed again Report Southern Blot

15. Fluorescent PCR Number of repeats = (242-151)/3 = ~30 rpts Number of repeats = ~ 21 rpts + ~ 66 rpts

16. 5.2kb 2.8kb Full mutation female Full mutation male Normal male Normal female Premutation female Premutation male Southern Blot – OX1.9 Probe Xq27.3 Ox1.9 EcoR I Eag I EcoR I Eag1 – methylation sensitive – i.e. won’t cut methylated DNA 5.2kb 2.8kb

17. Interesting Prenatal Case • SB – EcoRI and EagI, Ox1.9 • Cultured Amnio • Mother carrier of ~51 rpts (Int) and 129 rps (Pre) • Male fetus • Result – methylayed expanded allele, mosaicism for both an unmethylated expansion and a smaller fragment (deletion at fragile X locus). Clinical significance of mosaicism for a deletion uncertain

18. Alternative Testing Strategies • Asuragen FRAX PCR kit - Gene-specific FMR1 PCR and CGG Repeat Primed (RP) PCR(TP-PCR) • Reduce amount of Southern Blotting required – downsides lose familiarity/expertise, potentially reduced batch sizes leading to wasted capacity on blot, more expensive

19. References: • Filipovic-Sadic, S. et al. (2010) Clinical Chemistry, 56:3, 399-408. A novel FMR1 PCR Method for the Routine Detection of Low Abundance Expanded Alleles and Full Mutations in Fragile X Syndrome • Garcia-Arocena, D. and Hagerman, P.J. (2010) Human Molecular Genetics, 19, R83-R89. Advances in understanding the molecular basis of FXTAS • Tan, H. et al. (2009) Neuroscience Letters, 466, 103-108. RNA-mediated pathogenesis in fragile X-associated disorders. • Rodriguez-Revenga, L. et al. (2010) Neurology, 75, 1370 – 1376. Motor and mental dysfunction in mother-daughter transmitted FXTAS • Batra, R. et al. (2010) Human Molecular Genetics, 19, R77-R82. Partners in crime: bidirectional transcription in unstable microsatellite disease. • O’Rourke, J.R. and Swansin, M.S. (2009) The Journal of Biological Chemistry, 284, 7419-7423. Mechanisms of RNA-mediated Disease • Practice Guidelines for molecular diagnosis of Fragile X Syndrome – CMGS February 2005