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Home , Polysomy

Preimplantation Genetic Diagnosis (PGD)

AS3323/5621 Lecture 7 Sept 19, 2017

1 Risks of Fetal Loss

A total of 634,272 women and 1,221,546 pregnancy outcomes in Denmark from 1978 to 1992. 2 Anderson et al. BMJ, 2000,320:1708-1712. Meiotic Non-disjunction:% ?

3 P Q

Abnormal number of in a cell.

4 Aneuploidy: 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).

http://www.asrm.org/uploadedFiles/ASRM_Content/News_and_Publications/Practice_Guidelines/Commi ttee_Opinions/Preimplantation_genetic_testing(1).pdf 5 “Common” Types of Trisomy Trisomy 21 – Down’s Syndrome 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 47, XXY (), 1/1,000 males 47, XXX (super females), many un-diagnosed Polysomy X e.g., XXXX Trisomies of other chromosomes partial, and rare incompatible with life 6 • No other live births of full monosomy – abortion • Cancer cells (lymphocytes) • Only partial in live births

• Turner’s Syndrome: XO females

7 Why does aneuploidy lead to fetal death? dosage • Intolerance to deviations from diploid • Extra or missing chromosomes causes developmental defects and/or death (monosomy, trisomy)

• Sex chromosomes exception • On the surface 8 Central Dogma of

9 http://cnx.org/contents/Z7qBU2RZ@5/The-Central-Dogma-and-Basic-Tr 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

13 14 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, , – 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 http://www.youtube.com/watch?v=nm8Ai1CI9Is

AS 5621:needs to know procedure 20 FISH Probes

Locus specific / telomeric Centromeric Whole chromosome21 probes probes painting probes Interphase Chromatin, Metaphase Chromosomes

22 Chromatin are relatively condensed in blastomeres Aneuploidy Analysis with FISH

Nucleus Denaturation Biopsy Addition of fixation probes of probes/nucleus 13 = red 16 = aqua 18 = blue 21 = green 22 = yellow

Wash to eliminate Visualization Hybridization with proper filters non-specific hybridization (4 hrs / overnight) Wash off 22 13 probes Re-hybridize Y with X = yellow Y = aqua 16 18 15 = green 17 17 = orange 15 X 21

9 probes: X, Y, 13, 15, 16, 17, 18, 21, 22 23 Aneuploidy Analysis (Screening of 9 Chromosomes)

18 13 15 21 22

15 16 X 22 18 17

16 13 17

21 Y

1st FISH 2nd FISH 13 (red) 16 (aqua) 18 (blue) 21 (green) X (yellow) Y (aqua) 22 (gold) 15 (green) 17 (orange)

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 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 Chromosome Structure

31 Robertsonian Translocations

– Involve the acrocentric chromosomes (13, 14, 15, 21, and 22)

– The short arm is made up of satellite DNA, which is lost and the chromosomes join at their .

– Balanced Robertsonian translocation: two chromosomes are fused, giving rise to 45 chromosomes rather than 46.

– The most common translocation involves chromosomes 13 and 14,

1/1,300 32 The long arms of chromosomes 13 and 14 are fused, but no genetic material was lost - this person is completely normal despite the translocation (balanced). 33 Punnett Square:

Bb X Bb

What are the genotypes and phenotypes of the gametes and offspring ?

% of each genotype, % of each phenotype?

How about AaBb X AABB ? 50% offspring: Physically normal 25% carrier 25%: monosomy 25%: Down’s syndrome

Mother 14, 21 14, 21 14-21, 21 14, 0 14-21, 0

35 Reciprocal Translocations

– Reciprocal translocations, breaks occur in two chromosomes which re-join incorrectly. – Exchange of material between non-homologous chromosomes – Reciprocal translocations are found in about 1 in 600 newborns. – Usually harmless for carriers – Reproduction problems

36 Chromosomal translocation of the 4th and 20th chromosomes (HW 3 !!!)

• Balanced carriers normal

• Reproduction problems – Haploid – missing

37 PGD example #1

Chromosome Structural Analysis

• Balanced translocation • Deletion • Inversion

38 Cornell First Healthy Baby from PGD for Balanced Translocation • 27 year old (April, 1997). • During 1/95-9/96 (21 months) 5 spontaneous miscarriages & 1 ectopic pregnancy.

46,XX,t(4;11)(q21;q13) 46,XX (Amniocyte)

11 11 der(4) 4

der(11) PGD 11 4 4

WCP 4 WCP 11

A healthy girl was born in Dec, 1997.

39 Metaphase spreads: chromosomes, interphase: chromatin