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Fragile X Syndrome Mutation by Methylation Sensitive PCR
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Fragile X Syndrome Mutation by Methylation Sensitive PCR

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  • “ The objective of my project was to develop an efficient Dx test for Fragile X syndrome based on a later sited publication”
  • Most common cause of inherited mental retardation in males “ most common phenotypic characteristic…”
  • “ X-Inactive Specific Transcript” (XIST) XIST does not produce a protein - Causes chromosome inactivation Expression of XIST causes…
  • Low (but detectable) FMR-1 gene expression
  • By Formula: Would have to be less than 229 bp @ RU & less than 285 bp @ RM to have less 55 repeats capable of producing a PCR product Avg ~29-CGG = 151bp
  • In a clinical setting, will have to be performed with fluorescent markers using an ABI sequencer
  • Transcript

    • 1. Detection of Fragile X Syndrome Mutation using By: Connie Ross MS-PCR
    • 2. Characteristics
      • Elongated Face
      • Increased Head Circumference
      • Large Everted Ears
      • Prominent Jaw
      • High-Pitched Jocular Speech
      • Connective Tissues Disorders
      • Macroorchidism
      • Hypotonia
      • *Mild to Severe Intellectual Impairment
      • Generally Good Health & Normal Growth
    • 3. Behaviors
      • Hyperactivity
      • Aggressiveness
      • Attention Deficit
      • Hand Flapping
      • Aversion to Touch & Noise
      • Speech Disturbances
      • Poor Eye Contact
    • 4. Patient Management
      • Structured Environment
      • Individualized Educational Plans
      • Behavorial Medications
    • 5. Inheritance
      • X-Chromosome
      • Sex-Linked Semi-Dominate Inheritance
      • Mothers of children with Fragile-X are
      • carriers with either a pre or full mutation
        • Mutations must pass through a female to
        • have a phenotypic effect in offspring
      ~ 1: 4000-6000 Males ~ 1: 8000-9000 Females
    • 6. FMR1 Gene
      • Covers 39kb
      • Consists of 17 Exons
      • Within Exon 1 (in the 5’ untranslated region) is a CGG-triplet repeat
      ~230+ Full Mutation 55-230 PreMutation 45-54 Gray Zone 5-44 Normal Telomeric Region of the X-chromosome Xq27.3 - CH 3 Methylation is responsible for inactivation of the FMR1 gene
    • 7. Diagnostic Methods A good test must have the ability to determine (both) Expansion & Methylation
    • 8. XIST Gene as a Control FMR1 XIST FMR1 XIST FMR1 XIST Male Female Inactive Active XIST Functions as an Internal Standard & Control in the Promoter Region to Determine Methylation Its Methylation Pattern Opposes that of FMR-1 X Y X a X i CH 3
    • 9. Diagnostic Patterns
    • 10. Deamination Bisulfate +  Cytosine (unmethylated) Uracil
    • 11. BiSulfate Modification
      • Using primers specific for Converted & Unconverted DNA in PCR
        • allows determination of methylation status
      • Improves conditions for PCR
      UnMethylated DNA ggg g c g ga c c g c g… ggg g u g ga u u g u g… Methylated DNA ggg g c m g ga c c m g c m g… ggg g c m g ga u c m g c m g…
    • 12. Promoter Region (Methylation) Repeat Region (Number of CGG-repeats) MS-PCR Concept
    • 13. Promoter Region MS-PCR
    • 14. 100 bp marker Normal Female Normal Female Fragile X Female Neg. Ctrl Normal Male Fragile X Male 100 bp marker Promoter Region MS-PCR 100 bp 200 bp 300 bp 400 bp
    • 15. Re: Methylation Other Normal Samples Tested 100 bp marker Male - H0011 FM Std. - H0016 Male - H0012 Male - H0014 Neg. Ctrl 100 bp marker 100 bp marker Female - H0020 Female - H0021 Female - H0022 Female - H0023 Female - H0024 Female - H0025 FM Std. - H0016
    • 16. Repeat Region MS-PCR
    • 17. Repeat Region MS-PCR 100 bp marker Normal Female Normal Female Fragile X Female Neg. Ctrl Normal Male Fragile X Male 100 bp marker H0016 – ( 22 ) UnMethylated & ( 22 ) Methylated Repeats H0018 – ( 24 ) UnMethylated & ( 22 ) Methylated Repeats NA05855 – ( 32 ) Methylated Repeats H0010 – ( 38 ) UnMethylated Repeats NA06897 - >230 Repeats 100 bp 200 bp 300 bp 400 bp
    • 18. Re: #CGG-Repeats Testing of all Samples 100 bp marker Fragile-X Male H0010 H0011 H0012 H0014 Neg. Ctrl Fragile-X Female H0016 H0018 H0019 H0020 Neg. Ctrl. H0021 H0022 H0023 H0024 H0025 Neg. Ctrl. 100 bp marker
    • 19. Gold Standard Test Results Southern Blot 29 29 28 22 28 29 22 29 29 29 23 29 Conventional PCR
    • 20. Validation
    • 21. Bringing Research into Patient Care
    • 22. Acknowledgements Dr. Nader Ghebranious PhD Lynn Ivacic Chuck Dokken Dr. Philip Giampietro MD Dr. E. McPherson MD Christina Zaleski Steve Kaiser Dr. Mark Borchardt PhD Research Foundation Donors
    • 23. Work Cited
      • Maddalena, Anne, et al. “Technical Standards and Guidelines for Fragile X: The First of a Series of Disease-Specific Supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics.” Genetics IN Medicine 3.3 May/June (2001):
      • 200-204.
      • Weinhausel and Haas. “Evaluation of the Fragile X (FRAXA) Syndrome with Methylation-Sensitive
      • PCR.” Human Genetics 108 (2001): 450-458.