Fragile X Syndrome Mutation by Methylation Sensitive PCR
- 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
- 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
- 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
- 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.
Editor's Notes
- #2: “ The objective of my project was to develop an efficient Dx test for Fragile X syndrome based on a later sited publication”
- #3: Most common cause of inherited mental retardation in males “ most common phenotypic characteristic…”
- #9: “ X-Inactive Specific Transcript” (XIST) XIST does not produce a protein - Causes chromosome inactivation Expression of XIST causes…
- #15: Low (but detectable) FMR-1 gene expression
- #17: 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
- #18: In a clinical setting, will have to be performed with fluorescent markers using an ABI sequencer