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
  • Fragile X Syndrome Mutation by Methylation Sensitive PCR

    1. 1. Detection of Fragile X Syndrome Mutation using By: Connie Ross MS-PCR
    2. 2. Characteristics <ul><li>Elongated Face </li></ul><ul><li>Increased Head Circumference </li></ul><ul><li>Large Everted Ears </li></ul><ul><li>Prominent Jaw </li></ul><ul><li>High-Pitched Jocular Speech </li></ul><ul><li>Connective Tissues Disorders </li></ul><ul><li>Macroorchidism </li></ul><ul><li>Hypotonia </li></ul><ul><li>*Mild to Severe Intellectual Impairment </li></ul><ul><li>Generally Good Health & Normal Growth </li></ul>
    3. 3. Behaviors <ul><li>Hyperactivity </li></ul><ul><li>Aggressiveness </li></ul><ul><li>Attention Deficit </li></ul><ul><li>Hand Flapping </li></ul><ul><li>Aversion to Touch & Noise </li></ul><ul><li>Speech Disturbances </li></ul><ul><li>Poor Eye Contact </li></ul>
    4. 4. Patient Management <ul><li>Structured Environment </li></ul><ul><li>Individualized Educational Plans </li></ul><ul><li>Behavorial Medications </li></ul>
    5. 5. Inheritance <ul><li>X-Chromosome </li></ul><ul><li>Sex-Linked Semi-Dominate Inheritance </li></ul><ul><li>Mothers of children with Fragile-X are </li></ul><ul><li>carriers with either a pre or full mutation </li></ul><ul><ul><li>Mutations must pass through a female to </li></ul></ul><ul><ul><li>have a phenotypic effect in offspring </li></ul></ul>~ 1: 4000-6000 Males ~ 1: 8000-9000 Females
    6. 6. FMR1 Gene <ul><li>Covers 39kb </li></ul><ul><li>Consists of 17 Exons </li></ul><ul><li>Within Exon 1 (in the 5’ untranslated region) is a CGG-triplet repeat </li></ul>~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. 7. Diagnostic Methods A good test must have the ability to determine (both) Expansion & Methylation
    8. 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. 9. Diagnostic Patterns
    10. 10. Deamination Bisulfate +  Cytosine (unmethylated) Uracil
    11. 11. BiSulfate Modification <ul><li>Using primers specific for Converted & Unconverted DNA in PCR </li></ul><ul><ul><li>allows determination of methylation status </li></ul></ul><ul><li>Improves conditions for PCR </li></ul>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. 12. Promoter Region (Methylation) Repeat Region (Number of CGG-repeats) MS-PCR Concept
    13. 13. Promoter Region MS-PCR
    14. 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. 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. 16. Repeat Region MS-PCR
    17. 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. 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. 19. Gold Standard Test Results Southern Blot 29 29 28 22 28 29 22 29 29 29 23 29 Conventional PCR
    20. 20. Validation
    21. 21. Bringing Research into Patient Care
    22. 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. 23. Work Cited <ul><li>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): </li></ul><ul><li>200-204. </li></ul><ul><li>Weinhausel and Haas. “Evaluation of the Fragile X (FRAXA) Syndrome with Methylation-Sensitive </li></ul><ul><li>PCR.” Human Genetics 108 (2001): 450-458. </li></ul>