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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

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 detection for large FMR-1 expansion mutations in males and females

  • 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 detection for large FMR-1 expansion mutations in males and females

  • 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 detection for large FMR-1 expansion mutations in males and females

    • 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 detection for large FMR-1 expansion mutations in males and females

    mPCR

    mTP

    nmTP

    Normal female result


    Premutation male result
    Premutation male result detection for large FMR-1 expansion mutations in males and females

    nmPCR

    mPCR

    mTP

    nmTP


    Full mutation results

    mTP detection for large FMR-1 expansion mutations in males and females

    nmTP

    mTP

    nmTP

    Male full mutation (TP-PCR only)

    Female full mutation (TP-PCR only)

    Full mutation results


    Validation results summary
    Validation results summary detection for large FMR-1 expansion mutations in males and females

    • 119 samples tested

    • No false positive or negative results found

    • 3 samples had odd profiles


    Validation results summary1
    Validation results summary detection for large FMR-1 expansion mutations in males and females

    • 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 blots detection for large FMR-1 expansion mutations in males and femalesAudit 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 detection for large FMR-1 expansion mutations in males and females

    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 detection for large FMR-1 expansion mutations in males and females

    nmTP

    Prenatal samples

    • Eight of thirteen negative showed no expansion


    Prenatal samples2

    mTP detection for large FMR-1 expansion mutations in males and females

    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 detection for large FMR-1 expansion mutations in males and females

    • 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 detection for large FMR-1 expansion mutations in males and females

    • 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 detection for large FMR-1 expansion mutations in males and females

    • 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