Fragile X Syndrome

1. SP5 -Validation of Methylation Specific PCR for the detection for large FMR-1 expansion mutations in males and females Stacey Mutch East Midlands Regional Molecular Genetics Service

2. Fragile X Syndrome • Caused by inactivation of FMR-1 gene and a corresponding reduction of FMRP • Inactivation is caused by methylation, due to expansion of a triplet repeat in 5’UTR of gene • Normal range 6-49 repeats • Intermediate 50-58 repeats • Premutation range 59-200 repeats and unmethylated • Full mutation range >200 repeats and methylated • Laboratories receive a high number of referrals • A proportion require Southern blotting • Developed and validated a PCR based alternative

3. MS-PCR • Original MS-PCR developed by Zhou et al, has bisulphite modified DNA as a template, primers designed to the antisense DNA strand • Sizing PCR for unmethylated alleles • Sizing PCR for methylated alleles • TP-PCR for methylated alleles • Modified for capillary based analysis and to include a TP-PCR for unmethylated alleles • Technique been validated for use on postnatal samples and also tested on prenatal samples

4. Expected results • In males • Normal alleles – unmet • Premutation alleles – unmet • Full mutation alleles - met • In females, due to X chromosome inactivation • Normal alleles – both met and unmet • Premutation alleles – both met and unmet • Full mutation alleles – met only • Mosaicism is common in Fragile X • Methylated and unmethylated full mutation alleles • Premutation and full mutation alleles • Full mutation and normal alleles

5. nmPCR mPCR mTP nmTP Normal female result

6. Premutation male result nmPCR mPCR mTP nmTP

7. mTP nmTP mTP nmTP Male full mutation (TP-PCR only) Female full mutation (TP-PCR only) Full mutation results

8. Validation results summary • 119 samples tested • No false positive or negative results found • 3 samples had odd profiles

9. Validation results summary • 11/42 (26%) full mutation samples showed mosaicism (nmTP-PCR expansions) • No evidence of this seen on their Southern blots • Cannot distinguish between mosaic and premutation females • 119 samples tested • No false positive or negative results found • 3 samples had odd profiles

10. Reducing the need for Southern blotsAudit over 3 month period • 28 samples required Southern blotting, on 7 blots • Only 4 would have needed blotting after MS-PCR • Reduce down to 2 Southern blots • 3 samples required more DNA, 2 sufficient for MS-PCR

11. mTP nmTP Prenatal samples • Twenty three prenatal samples available to test • Ten positive, thirteen negative • All positive showed a clear expansion on one or both TP-PCRs

12. mTP nmTP Prenatal samples • Eight of thirteen negative showed no expansion

13. mTP mTP zoom nmTP nmTP zoom Prenatal samples • Five of the thirteen negatives showed some kind of expansion in one or both TP-PCRs

14. Prenatal samples • Maternal contamination by QF-PCR was performed • One found to have a very low level, just below the minimum level detectable by this assay • The remaining four showed possible low levels at one or more markers • A sensitivity experiment was designed using a positivesample(50,20,10,7.5,2.5,1%)mixedwith either a negative female or a negative male sample • Just 5% positive sample in females and 2.5% in males clearly showed an expansion in TP-PCRs • Even 1% appeared different to negative alone

15. Summary • MS-PCR developed for Fragile X testing • Methylated and unmethylated sizing PCR • Methylated and unmethylated TP-PCR • Identifies • Male normal, premutation, full mutation and mosaics • Female normal from expansion • Predicted to reduce Southern blotting requirement • Prenatal samples can be tested, but method is highly sensitive and affected by very low levels of maternal contamination

16. References and acknowledgements • Zhou Y et al (2004) Robust fragile X (CGG)n genotype classification using a methylation specific triple PCR assay. J Med Genet. 41, e45. • I would like to thank all of the East Midlands Regional Laboratory staff for their help and assistance in the initial development of this assay and throughout my project