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. Normal female resultnmPCRmPCRmTPnmTP

6. Premutation male result nmPCRmPCRmTPnmTP

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

8. Validation results summary  119 samples tested  No false positive or negative results found  3 samples had odd profiles MaleFemale Normal814 Pre mutation 640 Full Mutation 1626 Mosaic81 Total3881

9. Validation results summary  119 samples tested  No false positive or negative results found  3 samples had odd profiles MaleFemale Normal814 Pre mutation 640 Full Mutation 16 6 (38%) 26 5 (19%) Mosaic81 Total3881   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

10. Reducing the need for Southern blots Audit 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 Sample typeNumberMS-PCR sufficientBlotting required F Normal20Yes0 F Premutation3No3 F Full mutation1No1 M Intermediate1Yes0 M Premutation1Yes0 M Klinefelter2Yes0 Total284

11. 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 mTPnmTP

12. Prenatal samples  Eight of thirteen negative showed no expansion mTPnmTP

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

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 positive sample (50, 20, 10, 7.5, 2.5, 1%) mixed with 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