This document summarizes the history and current state of preimplantation genetic diagnosis (PGD). PGD involves in vitro fertilization, embryo biopsy, and genetic testing to select embryos without inherited diseases or chromosomal abnormalities for transfer. Over 20 years, PGD has expanded from sex selection to screening for over 100 single gene defects, chromosomal abnormalities, HLA matching, and assessing risk for multifactorial diseases. New techniques like array comparative genomic hybridization and single nucleotide polymorphism analysis with karyomapping now allow comprehensive chromosome and single gene defect screening, identification of aneuploidies, duplications/deletions, and determination of parental origins.

1. Gabbay Award Lecture
Brandeis University
November 16th, 2009
‘Designer babies’:
twenty years on
Alan H Handyside
London Bridge Fertility, Gynaecology
And Genetics Centre
London
United Kingdom

3. Preimplantation Genetic Diagnosis (PGD)
• In Vitro Fertilisation (IVF)
Downregulation of ovarian
folliculogenesis
Superovulation and egg collection
Intracytoplasmic sperm
microinjection (ICSI) and embryo
culture
Cleavage stage biopsy by zona
drilling and micromanipulation
• Single cell genetic analysis (12-72h)
• Selective transfer of unaffected
embryos 3-5 days post fertilisation

4. Pregnancies from biopsied human
preimplantation embryos sexed by Y-specific
amplification
Handyside et al Nature (1990) 344, 768
Born July, 1990
Gender identification
and selective transfer of
unaffected female
embryos in X-linked
disease
Amplification of DYZ1
using 40 cycles of PCR
from single cleavage
stage blastomeres

5. Inherited conditions diagnosed by PGD
Familial polyposis coli
Retinoblastoma
Li-Fraumeni syndrome
Chromosomal
abnormalities
Aneuploidy
Translocations
Structural
Other
HLA matching
Single gene defects
Duchenne muscular
dystrophy
Haemophilia
Adrenoleukodystrophy
Cystic fibrosis
Spinal muscular atrophy
Beta-thalassaemia
Sickle cell disease
Tay-Sachs disease
Gaucher disease
Huntington’s disease
Myotonic dystrophy

6. Strategies for single cell genetic analysis
SINGLE OR MULTIPLE CELL BIOPSY LYSIS
A B C D E
NESTED PCR
OUTER PCR MULTIPLEX F-PCR MULTIPLEX PCR PEP/DOP-PCR
INNER PCR
HETERODUPLEX
FORMATION
PAGE
MDA
AUTOMATED
SEQUENCER
SEQUENCING
MINISEQUENCING
AUTOMATED
SEQUENCER
MULTIPLEX PCR
SEQUENCING
MINISEQUENCING
AUTOMATED
SEQUENCER
ANY COMBINATION
OF CONVENTIONAL
DNA BASED TESTS
CGH
MICROARRAYS
CGH
DNA free reagents
and conditions

7. Fiorentino et al (2005) Eur J Hum Genet 13, 953

8. Handyside et al (2004) Mol Hum Reprod 10, 167

9. Locus 1 cell 2 cells 5 cells
Chromosome 7 CFTR exon 10 and intragenic marker (PCR and F-PCR)
CF ΔF508
97/100 2 2 2 1 2 1 2 2 2 1 2 2 2 2 1
CF ΔF508
97/100 1 2 2 2 2 1 2 2 1 2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 2
CF ΔF508
148/151 1 2 2 2 1 2 2 1 2 2 2 2 1 2 2 2 2 1 2 2 1 1
CF 17βTA
247/251 2 2 2 1 1 1 2 1 2 2 1 1 1 2 1 1 1 2 1 2 1
Chromosome 6 STR markers for HLA matching (F-PCR)
D6S276
205/221 2 1 2 2 2 1 2 1 2 1 1 1 1 2 2 1 1
MIB
180/184 2 2 1 2 2 2 2 2 1 1 1 2 2 1 1 1 1 1 1 2 1 2 1 2
D6S291
157/163 2 2 2 1 2 2 1 2 2 1 1 2 1 2 1 1 1 2 1 1
HLABC
114/144 1 2 1 1 2 2 1 1 1 1 1 1 1 2 1 1 1 1
D6S265
113/121 1 1 1 1 2 2 1 1 2 2 1 2 2 1 2 1 1 2 1
Heterozygous Allele dropout Amp failure
1
2
Allele specific preferential amplification

10. Preimplantation genetic haplotyping following
MDA from single cells
Renwick et al (2006) Reprod Biomed Online 13, 110

11. Embryo 2
Monosomy 16, 21
Trisomy 22

12. Chromosome imbalance and
genotyping by microarrays
•DNA microarrays with probes spaced
typically at 1 Mb intervals genome wide for
comparative genomic hybridisation
(array CGH)
• High density genome wide single
nucleotide polymorphism (SNP) genotyping
arrays

13. CONTROL TEST

14. CONTROL TEST
1.5
1.0
0.5

15. 1.5
1.0
0.5
CONTROL TEST

16. 1.5
1.0
0.5
CONTROL TEST

17. First or first and second polar
body biopsy on day 0 or day 1
Array comparative
genomic hybridisation
(CGH) following
whole genome
amplification

18. Single nucleotide
polymorphisms (SNPs)
• 10 million SNPs across
human genome
• Many biallelic (AA, AB, BB)
• Major contribution to
genetic diversity, inherited
disease and variants
associated with common
multifactorial conditions

19. Karyomapping
• A universal method for genome wide analysis of
genetic disease based on mapping crossovers
between parental haplotypes
• High density genome wide single nucleotide
polymorphisms (SNP) genotyping of proband, parents and
appropriate family member(s) to establish phase (Illumina
Human CNV 370 Infinium-II Quad and Duo >300K SNP loci)
• Mendelian analysis and karyomapping of the parental and
grandparental haplotypes for each chromosome or
chromosome segment in recombinant chromosomes
Handyside et al (2009) J Med Genet Online First

20. ♂ ♀
BAA
ABA
AAB
BBA
AAB
ABA
AAB
AAB

21. A ♂ ♀
B
C D
Karyomapping combines
genome wide linkage based
detection of single gene defects
(A) with chromosomal
aneuploidy including
monosomy/deletions (B) and
trisomies involving inheritance of
two different meiotic
chromosomes from one parent
(D). Chromosome duplication is
not detected (C).

22. Family 1

23. Family 2
Preimplantation genetic diagnosis
for cystic fibrosis
Whole genome amplification by
isothermal multiple displacement
amplification of 2-10 cells in each
biopsy/embryo

24. Embryo 7
13, 16, 18, 21, 22
Monosomy 18, 21, 22
X, Y, 21
Trisomy X
Monosomy 21
Array CGH
Karyomap

25. Mutation
detection
Multiplex PCR and
minisequencing
Preimplantation
genetic haplotyping
Fluorescence
in situ
hybridisation
Array CGH
Quantitative SNP array analysis
and
Karyomapping
Single gene defects
Single or combination  Any  Any
Exclusion    
SGD + HLA typing    
Chromosome screening
Aneuploidy  (5-12 chr) (24 chr) (24 chr)
Meiotic aneuploidy (PB only) (PB only)  Trisomy only
With parental origin    
With meiotic origin    
Mosaicism   
Duplications/deletions   
Uniparental disomy   
Translocation chromosome imbalance
Reciprocal/Robertsonian    
Normal vs balanced    
With 24 chr aneuploidy    
Other
Multifactorial recurrence risk 
Copy Number Variants 
Errors
Allele dropout   
Contamination   

26. SNP analysis and Karyomapping
• A universal method for genome wide analysis of genetic defects
and their parental origin
• Complementary to standard methods of SNP data analysis for
molecular cytogenetics
• Parental origin of aneuploidies and meiotic phase of origin of
trisomies identified
• Inheritance and parental origin of copy number variants and
structural chromosomal abnormalities can be analysed
• Enhanced linkage based analysis of loci genome wide for
diagnosis of single gene defects and analysis of recurrence risk of
multifactorial conditions
• Applicable at the single cell level for preimplantation genetic
diagnosis (PGD)

27. Acknowledgements
Genoma Laboratory, Rome
Anil Biricik
Francesco Fiorentino
Bridge Genoma, London
Helen Tempest
Mira Grigorova
Alan Thornhill
Department of Biosciences,
University of Kent
Alem Gebrasselasi
Darren Griffin
BlueGnome, Cambridge
Anthony Brown
Tony Gordon
Graham Snudden
Nick Haan
Department of Pathology,
University of Cambridge
Ben Dunmore
Nabeel Affara
Genetics and IVF Institute,
Fairfax, Virginia
Gary Harton
Brian Mariani