slide1
Download
Skip this Video
Download Presentation
Fragile X Syndrome

Loading in 2 Seconds...

play fullscreen
1 / 16

Fragile X Syndrome - PowerPoint PPT Presentation


  • 96 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' Fragile X Syndrome' - yonah


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

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
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
ad