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Hutchinson-Guilford Progeria

Hutchinson-Guilford Progeria. premature aging lifespan = 13.4 years retarded growth midface hypoplasia micrognathia alopecia low adiposity osteodysplasia premature, severe atherosclerosis -death due to MI. De Sandre-Ciovannoli, Science express, 17 April 2003. Lamin A mutations in HGS.

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Hutchinson-Guilford Progeria

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  1. Hutchinson-Guilford Progeria • premature aging • lifespan = 13.4 years • retarded growth • midface hypoplasia • micrognathia • alopecia • low adiposity • osteodysplasia • premature, severe • atherosclerosis • -death due to MI De Sandre-Ciovannoli, Science express, 17 April 2003

  2. Lamin A mutations in HGS Exons 11 and 12 code the Lamin A tail (not lamin c) Red is coiled-coil and blue is globular domains 1824C>T is aa conservative (G608G) but - in 300 con. 1824C>T creates a cryptic donor site at 1819, -50 aa del

  3. Best guess Most diseases are probably interactionsbetween polygenic heritable events, and environmental pressures leading to somatic epigenetic changes. Translation: diseases are complicated.

  4. Gene by Environment Interaction Predisposition Event Disease DNA FAP MSH BRCA LDLr hydrocarbons radiation estrogens low fiber colon CA colon CA breast CA atherosclerosis

  5. Microarrays-the big net. Ideal disease-hunter: genomic scale protein quantitation and sequencing. Imperfect solution A: genomic scale detection of mRNA level. Problem: little information on protein level Imperfect solution B: genome-wide SNP/haplotype. Problem: statistical limits on patient populations Common compromise: microarray profiling mRNA transcripts (transcript profiling) to identity target areas. Target genes are then followed by proteomics and SNPs.

  6. Array flavors DNA detection (SNP, genotyping, etc.) • short oligonucleotides to detect mismatches RNA detection (transcript profiling) • Plasmid • Inserts • Long oligonucleotides (60 mers) • Short oligonucleotides (20 mers)

  7. Hybridization-basic elements • Hybridization = Annealing - Melting • CRUCIAL: non-covalent, hydrogen bonds • -->equilibrium rules, binding is statistical • Best hybridization occurs with: • long sequences (no hyb when nt<4) • high salt concentration (hybrids melt in water) • low temperatures (hybrids melt with heat) • G and C (3 H) bind better than A and T (2 H) • self-complementarity is low (high GC is bad)

  8. Base-pairing (the stuff of life) A T G C T A C G Lewin. Genes VII page 8.

  9. Tm-a good thing. Tm is a measure of the stability of DS-DNA under a given set of conditions. Stability, and therefore Tm, is affected by: Strand length - the longer the strand, the higher the Tm Base Composition - higher the GC content, the higher the Tm. Ionic Strength - as the ionic strength increases, so does Tm. Double helical DNA is stabilised by cations. Divalent cations (eg Mg2+) are more effective than monovalent cations (+ or K+). Organic Solvents - formamide for instance lowers the Tm by weakening the hydrophobic interactions.

  10. Melting Curves-Tm measured Tm Tm

  11. PCR Primer design www.oligo.net

  12. Array Choice Factors Expression profiling: Sequence known? Not known? Oligo arrays cDNA arrays High confidence Clone drift/cross hyb Immediate ID sequence clones

  13. Sample selection • isolate the purest phenotypic examples of test and control • laser capture microdissection (LCM) • always control for treatment and manipulation • people are the most meaningful, but least controllable • animals are highly controllable, but less meaningful • cell systems (in vitro) are controlled, but meaningful? • small amounts of RNA can be amplified • while purifying cells is good, the processing is bad. • The quality of the results are directly proportional to the samples that are chosen.

  14. Laser Capture Microdissection

  15. The importance of purity Human colon cancer Blue are normal cells Red are tumor cells

  16. Assessing sample quality • Amount > 5 ug total RNA or 500 ng of poly A+ • Basic: O.D. 260/280 ratio >2.1, • nucleic acids absorb at 260, protein at 280 nm • thus, increasing impurity reduces ratio • Better: agarose gel electrophoresis, EtBR stained • if total RNA, 28s = 2 x 18s ribosomal (Lab-on-chip) • or • Q-PCR of a low and high gene, against standard • Best: test chip

  17. * * Hybridized Probe Cell * * * Single stranded, labeled RNA target Oligonucleotide probe Millions of copies of a specific oligonucleotide probe GeneChip® Probe Arrays GeneChipProbe Array 11 µm 1.28cm >1 million probes Image of Hybridized Probe Array

  18. Light (deprotection) Mask TTOOO OOOOO HO HO OOO T – Substrate Light (deprotection) Mask CATAT AGCTG TTCCG TTCCO TTOOO C – Substrate REPEAT Synthesis of Ordered Oligonucleotide Arrays

  19. 3´ Multiple oligo probes GeneChip®Expression Array Design Gene Sequence Probes designed to be Perfect Match Probes designed to be Mismatch

  20. Procedures for Target Preparation Cells Labeled transcript AAAA IVT (Biotin-UTP Biotin-CTP) L L L L Poly (A)+ RNA cDNA Fragment (heat, Mg2+) L L Wash & Stain Hybridize (16 hours) L L Scan Labeled fragments Streptavidin-Phycoerythrin (SAPE) Fluorescent stain-laser stimulated

  21. Analysis of expression level from probe sets A single, contiguous gene set for the rat B-actin gene. Each pixel is quantitated and integrated for each oligo feature (range 0-25,000) Perfect Match (PM) Mis Match (MM) Control PM - MM = difference score All significant difference scores are averaged to create “average difference” = expression level of the gene.

  22. Affymetrix® Instrument System Platform for GeneChip® Probe Arrays • Integrated • Exportable • Easy to use • Versatile

  23. GeneChip analysis of human atherosclerosis Dissect normal media from atherosclerotic lesion Prepare highly purified RNA O.D. 260/280 = 2.0 Reverse transcribe w/poly dT + T7 = cDNA Transcribe with T7 + biotin dUTP = cRNA Purify probe/hybridize to chip Wash and detect with avidin/PE + ab amplification Read fluorescent label And deconvolve genes

  24. Basic Bioinformatics-Scatterplot

  25. Transcript profiling of aged rat aorta. Affymetrix GeneChip analysis of 10 aortas @ 20 mo. vs. 3 mo.

  26. FAQs: How many replicates?

  27. Simple fold changes • Crude, insensitive--but effective Criteria: Present 1.5-fold up/down

  28. Hierachical clustering

  29. Statistical testing and ontology

  30. Pathways of genetic information

  31. Expression of Egr-1 mRNA in human lesions.

  32. Egr-1 mRNA and protein in lesions vs normal cells. Western blot A) B) Media E197 E221 E240 E243 20 Lesion M L M L M L M L 15 Egr-1 Egr-1 mRNA x x 10 5 Actin 0 E197 E196

  33. Expression screening by GeneChip • each oligo sequence (20 mer) is synthesized as a 11 µ square (feature) • each feature contains > 1 million copies of the oligo • scanner resolution is about 2 µ (pixel) • each gene is quantitated by 11 oligos and compared to equal # of mismatched controls • 44,000 genes are evaluated with 11 matching oligos and 11 mismatched oligos = 4 x 106 features/chip • features are photolithographically synthesized onto a 2 x 2 cm glass substrate

  34. GeneChip® Array Advantages – Specificity Oligo arrays cDNA arrays Gene “on” ~ 150 µm 24 µm Gene “off” Detection Pattern Single Spot

  35. Limitations to all microarrays. • dynamic range of gene expression: • very difficult to simultaneously detect low and high • abundance genes accurately • - each gene has multiple splice variants • 2 splice variants may have opposite effects (i.e. trk) • arrays can be designed for splicing, but complexity ^ 5X • - translational efficiency is a regulated process: • mRNA level does not correlate with protein level • - proteins are modified post-translationally • glycosylation, phosphorylation, etc. • - pathogens might have little ‘genomic’ effect

  36. Lipoprotein genes/variants Heart failure predictors Atherosclerosis markers Restenosis markers Coagulation factors Stress markers Inflammatory markers Infectious agents CardioChip in silico workup

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