‘Designer babies’:twenty years on
Alan H Handyside
London Bridge Fertility, GynaecologyAnd Genetics Centre
LondonUnited Kingdom
Gabbay Award LectureBrandeis University
November 16th, 2009
• In Vitro Fertilisation (IVF)Downregulation of ovarian
folliculogenesisSuperovulation and egg collectionIntracytoplasmic 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
Preimplantation Genetic Diagnosis (PGD)
Handyside et al Nature (1990) 344, 768
Pregnancies from biopsied human
preimplantation embryos sexed by Y-specific
amplification
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
Inherited conditions diagnosed by PGD
Familial polyposis coliRetinoblastomaLi-Fraumeni syndrome
Chromosomal abnormalities
AneuploidyTranslocationsStructural
OtherHLA matching
Single gene defects
Duchenne muscular dystrophy
HaemophiliaAdrenoleukodystrophy
Cystic fibrosisSpinal muscular atrophyBeta-thalassaemiaSickle cell diseaseTay-Sachs diseaseGaucher disease
Huntington’s diseaseMyotonic dystrophy
SINGLE OR MULTIPLE CELL BIOPSY LYSIS
NESTED PCR
OUTER PCR
MULTIPLEX F-PCR
MULTIPLEX PCR PEP/DOP-PCR
INNER PCR
HETERODUPLEXFORMATION
PAGE
MDA
AUTOMATEDSEQUENCER
SEQUENCINGMINISEQUENCING
AUTOMATEDSEQUENCER
SEQUENCINGMINISEQUENCING
AUTOMATEDSEQUENCER
MULTIPLEX PCR
ANY COMBINATION
OF CONVENTIONAL
DNA BASED TESTS
CGH
MICROARRAYS
A B C D E
CGH
DNA free reagents and conditions
Strategies for single cell genetic analysis
Locus 1 cell 2 cells 5 cells
Chromosome 7 CFTR exon 10 and intragenic marker (PCR and F-PCR)CF ΔF508
97/1002 2 2 1 2 1 2 2 2 1 2 2 2 2 1
CF ΔF50897/100
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 ΔF508148/151
1 2 2 2 1 2 2 1 2 2 2 2 1 2 2 2 2 1 2 2 1 1
CF 17βTA247/251
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)D6S276205/221
2 1 2 2 2 1 2 1 2 1 1 1 1 2 2 1 1
MIB180/184
2 2 1 2 2 2 2 2 1 1 1 2 2 1 1 1 1 1 1 2 1 2 1 2
D6S291157/163
2 2 2 1 2 2 1 2 2 1 1 2 1 2 1 1 1 2 1 1
HLABC114/144
1 2 1 1 2 2 1 1 1 1 1 1 1 2 1 1 1 1
D6S265113/121
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
Preimplantation genetic haplotyping following MDA from single cells
Renwick et al (2006) Reprod Biomed Online 13, 110
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
First or first and second polar body biopsy on day 0 or day 1
Array comparative genomic hybridisation (CGH) following whole genome amplification
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
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
B
C D
♂ ♀A
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).
Family 2Preimplantation genetic diagnosis
for cystic fibrosis
Whole genome amplification by isothermal multiple displacementamplification of 2-10 cells in eachbiopsy/embryo
Mutation detection
Multiplex PCR and minisequencing
Preimplantation genetic haplotyping
Fluorescence in situ
hybridisationArray 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
• 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)
SNP analysis and Karyomapping
Genoma Laboratory, RomeAnil BiricikFrancesco Fiorentino
Bridge Genoma, LondonHelen TempestMira GrigorovaAlan Thornhill
Department of Biosciences, University of Kent
Alem GebrasselasiDarren Griffin
BlueGnome, CambridgeAnthony BrownTony GordonGraham SnuddenNick Haan
Department of Pathology, University of Cambridge
Ben DunmoreNabeel Affara
Genetics and IVF Institute,Fairfax, Virginia
Gary HartonBrian Mariani
Acknowledgements