Advances in Preimplantation Genetic Diagnosis and Genome Analysis
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
2.
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
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
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
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
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).
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
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