1

Preimplantation Genetic Diagnosis

(PGD)

AS3323/5621

Lecture 7

Sept 19, 2017

2

Risks of Fetal Loss

A 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?

3

Aneuploidy

Abnormal number

of chromosomes in

a cell.

4

P

Q

centromere

5

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

Aneuploidy: the most common cause for early

pregnancy failure

6

“Common” Types of TrisomyTrisomy 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

Monosomy

• No other live births of full monosomy

– abortion

• Cancer cells (lymphocytes)

• Only partial monosomies in live births

• Turner’s Syndrome: XO females

7

8

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

Why does aneuploidy lead to fetal

death?

Central Dogma of Gene Expression

9http://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 ?

XYXX

Gene Dosage Problem ?

XYX-

AA

13

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

14

15

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.

16

– 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

for Aneuploidy: a committee opinion, March 2016

PGD Molecular Techniques

17

Single Cell Genetic Analysis

Pre-implantation Genetic Diagnosis is a technique based on single

cell genetic analysis at the chromosomal or nucleotide level.

18

Major Categories Of PGD1. Chromosome number: Aneuploidy

2. Chromosome structure– Translocation, deletion,

– duplication, inversion

3. Single gene disorder

4. X-linked disorders

5. Social sexing

19

PGD Molecular Methods

• FISH: Fluorescence In Situ

Hybridization

• PCR: Polymerase Chain Reaction

(see PGD example #2)

20

Fluorescence In Situ Hybridization

AS 5621:needs to know procedure

http://www.youtube.com/watch?v=nm8Ai1CI9Is

21

FISH Probes

Locus specific / telomeric

probes

Centromeric

probes

Whole chromosome

painting probes

22

Interphase Chromatin, Metaphase

Chromosomes

Chromatin are relatively condensed in blastomeres

23

Aneuploidy Analysis with FISH

BiopsyNucleus

fixationAddition of

probes

13 = red

16 = aqua

18 = blue

21 = green

22 = yellow

Denaturation

of probes/nucleus

Hybridization

(4 hrs / overnight)

Wash to eliminate

non-specific

hybridization

Visualization

with proper filters

Re-hybridize

with

X = yellow

Y = aqua

15 = green

17 = orange

22

16

13

17

Y

15

9 probes: X, Y, 13, 15, 16, 17, 18, 21, 22

21

18

X

Wash off

probes

24

Y

X

17

17

15

15

Aneuploidy Analysis

(Screening of 9 Chromosomes)

1st FISH

13 (red) 16 (aqua)

18 (blue) 21 (green)

22 (gold)

2nd FISH

X (yellow) Y (aqua)

15 (green) 17 (orange)

13

13

16

16

18

18

21

21

22

22

XY, 13/13, 15/15, 16/16, 17/17, 18/18, 21/21, 22/22

Problem of this approach?

25

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.

26

Trisomy 21 due to abnormal cell division

27

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.

Maternal Age and Trisomy 21

28

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.

29

Chromosome Structural Analysis

• Balanced translocation

• Deletion

• Inversion

30

Chromosome Translocations

• Robertsonian translocations

• Reciprocal translocations

• When balanced, carriers are

normal

Chromosome Structure

31

32

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 centromeres.

– 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

33

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).

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 ?

35

50% offspring:

Physically normal

25% carrier

25%: monosomy

25%: Down’s syndrome

Mother

14, 21

14-21, 21

14, 0

14-21, 0

Father

14, 21

36

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

37

Chromosomal translocation of the 4th

and 20th chromosomes (HW 3 !!!)

• Balanced carriers normal

• Reproduction problems– Haploid

– Genes missing

38

PGD example #1

Chromosome Structural Analysis

• Balanced translocation

• Deletion

• Inversion

39

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.

A healthy girl was born in Dec, 1997.

WCP 4 WCP 11

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

4

4

11

11PGD

4

11

der(11)

der(4)

Metaphase spreads: chromosomes, interphase: chromatin