Preimplantation Genetic Diagnosis (PGD)

Preimplantation Genetic Diagnosis (PGD) AS3323/5621 Lecture 7 Sept 19, 2017 1Risks of Fetal Loss 2A total of 634,272 women and 1,221,546 pregnancy outcomes in Denmark from 1978 to 1992. Anderson et al. BMJ, 2000,320:1708-1712. Meiotic Non-disjunction:% Trisomy? 3Aneuploidy Pcentromere QAbnormal number of chromosomes in a cell. 4Aneuploidy: the most common cause for early pregnancy failure Prevalence of oocyte and embryo aneuploidy increases with maternal age Also increase in chromosomally normal couples with recurrent early pregnancy loss or repeated failed IVF cycles despite the transfer of high-quality embryos (based on morphology). <a href="/goto?url=http://www.asrm.org/uploadedFiles/ASRM_Content/News_and_Publications/Practice_Guidelines/Commi" target="_blank">http://www.asrm.org/uploadedFiles/ASRM_Content/News_and_Publications/Practice_Guidelines/Commi </a>ttee_Opinions/Preimplantation_genetic_testing(1).pdf 5“Common” Types of Trisomy Trisomy 21 – Down’s Syndrome karyotype 47, XX +21 or 47, XY + 21 frequency ~ 1/600 births Trisomy 18 – Edward’s Syndrome karyotype 47, XX + 18 or 47, XY + 18 Frequency ~ 1 in 10,000 births Sex chromosome trisomies 47, XXY (Klinefelter Syndrome), 1/1,000 males 47, XXX (super females), many un-diagnosed Polysomy X e.g., XXXX Trisomies of other chromosomes partial, mosaic and rare incompatible with life 6Monosomy • No other live births of full monosomy – abortion • Cancer cells (lymphocytes) • Only partial monosomies in live births • Turner’s Syndrome: XO females 7Why does aneuploidy lead to fetal death? Gene dosage • Intolerance to deviations from diploid • Extra or missing chromosomes causes developmental defects and/or death (monosomy, trisomy) • Sex chromosomes exception • On the surface 8Central Dogma of Gene Expression 9<a href="/goto?url=http://cnx.org/contents/Z7qBU2RZ@5/The-Central-Dogma-and-Basic-Tr" target="_blank">http://cnx.org/contents/Z7qBU2RZ@5/The-Central-Dogma-and-Basic-Tr </a>Gene copy number vs. Dosage Two copies, two doses One copy, two doses 10 Gene Dosage Problem ? XX XY Gene Dosage Problem ? AA X- XY Basic Steps of IVF-PGD 1. Regular IVF (can be for fertile couples) 2. Embryo biopsy: day 3 (what cell stage?) 3. PGD by cellular/molecular analyses 4. Determine which embryos to transfer <ul style="display: flex;"><li style="flex:1">13 </li><li style="flex:1">14 </li></ul>Potency of Early Cleavage Stage Blastomeres (animal model) The world's first identical quadruplet bulls produced from a single four-cell embryo by in vitro fertilization were born at the University of Guelph in the fall of 1992. Johnson et al. Veterinary Record 1995, 137:15-16. 15 PGD Molecular Techniques – fluorescence in situ hybridization (FISH) – comparative genomic hybridization (CGH) – array CGH (aCGH) – digital polymerase chain reaction (dPCR) – single-nucleotide polymorphism (SNP) array – real-time quantitative PCR (qPCR) – next-generation sequencing (NGS). ASRM Practice Committee Brief Communication on Pre-Implantation Genetic Screening 16 for Aneuploidy: a committee opinion, March 2016 Single Cell Genetic Analysis Pre-implantation Genetic Diagnosis is a technique based on single cell genetic analysis at the chromosomal or nucleotide level. 17 Major Categories Of PGD 1. Chromosome number: Aneuploidy 2. Chromosome structure – Translocation, deletion, – duplication, inversion 3. Single gene disorder 4. X-linked disorders 5. Social sexing 18 PGD Molecular Methods • FISH: Fluorescence In Situ Hybridization • PCR: Polymerase Chain Reaction (see PGD example #2) 19 Fluorescence In Situ Hybridization <a href="/goto?url=http://www.youtube.com/watch?v=nm8Ai1CI9Is" target="_blank">http://www.youtube.com/watch?v=nm8Ai1CI9Is </a>20 AS 5621:needs to know procedure FISH Probes Centromeric probes 21 Locus specific / telomeric probes Whole chromosome painting probes Interphase Chromatin, Metaphase Chromosomes 22 Chromatin are relatively condensed in blastomeres Aneuploidy Analysis with FISH Nucleus fixation Denaturation of probes/nucleus Addition of probes Biopsy 13 = red 16 = aqua 18 = blue 21 = green 22 = yellow Wash to eliminate non-specific hybridization Visualization with proper filters Hybridization (4 hrs / overnight) Wash off probes <ul style="display: flex;"><li style="flex:1">22 </li><li style="flex:1">13 </li></ul>YRe-hybridize with X = yellow Y = aqua 15 = green 17 = orange <ul style="display: flex;"><li style="flex:1">16 </li><li style="flex:1">17 </li></ul>18 15 X 21 23 9 probes: X, Y, 13, 15, 16, 17, 18, 21, 22 Aneuploidy Analysis (Screening of 9 Chromosomes) 18 13 15 21 22 15 16 X 22 18 17 17 16 13 21 Y1st FISH 2nd FISH X (yellow) Y (aqua) 15 (green) 17 (orange) 13 (red) 16 (aqua) 18 (blue) 21 (green) 22 (gold) XY, 13/13, 15/15, 16/16, 17/17, 18/18, 21/21, 22/22 24 Problem of this approach? Chromosome Numerical Evaluation Currently the Major Indication for PGD •Avoid live-births with chromosomal disorders (eg, Down, Turner (XO), etc) •Reduce risks of miscarriages •Increase pregnancy rates? •Not with FISH alone •With new methods, yes !!! •Prior believe: common aneuploidy (13, 16, 18, 21, 22, X and Y), but later found aneuploidy can happen all chromosomes. 25 Trisomy 21 due to abnormal cell division 26 Down’s Syndrome • Impaired learning and physical growth, and a recognizable facial appearance usually identified at birth • Trisomy 21, accounts for 95% of Down’s Syndrome • Most, 88% 2 copies of maternal 21 • Down’s syndrome is caused in a minority (5% or less) of cases by a Robertsonian translocation of about a third of chromosome 21 onto chromosome 14. 27 Maternal Age and Trisomy 21 Eve fertile women should be careful when reproducing at an older age Michelle Duggar (50): 17 births (2 sets of twins) in 21 years, ~15 month/birth; last a premie born in 2009 at 42; miscarriage in 2011 at 44. 28 Chromosome Structural Analysis • Balanced translocation • Deletion • Inversion 29 Chromosome Translocations • Robertsonian translocations • Reciprocal translocations • When balanced, carriers are normal 30

Preimplantation Genetic Diagnosis (PGD)

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