SP5 -Validation of Methylation Specific PCR for the detection for large FMR-1 expansion mutations in...
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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 . Fragile X Syndrome. Caused by inactivation of FMR-1 gene and a corresponding reduction of FMRP

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Fragile X Syndrome

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Fragile x syndrome

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


Fragile x syndrome

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


Ms pcr

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


  • Expected results

    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


  • Normal female result

    nmPCR

    mPCR

    mTP

    nmTP

    Normal female result


    Premutation male result

    Premutation male result

    nmPCR

    mPCR

    mTP

    nmTP


    Full mutation results

    mTP

    nmTP

    mTP

    nmTP

    Male full mutation (TP-PCR only)

    Female full mutation (TP-PCR only)

    Full mutation results


    Validation results summary

    Validation results summary

    • 119 samples tested

    • No false positive or negative results found

    • 3 samples had odd profiles


    Validation results summary1

    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


    Reducing the need for southern blots audit over 3 month period

    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


    Prenatal samples

    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


    Prenatal samples1

    mTP

    nmTP

    Prenatal samples

    • Eight of thirteen negative showed no expansion


    Prenatal samples2

    mTP

    mTP zoom

    nmTP

    nmTP zoom

    Prenatal samples

    • Five of the thirteen negatives showed some kind of expansion in one or both TP-PCRs


    Prenatal samples3

    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


    Summary

    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


  • References and acknowledgements

    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


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