‘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

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

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

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

Embryo 2

Monosomy 16, 21

Trisomy 22

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

CONTROL TEST

CONTROL TEST

1.0

1.5

0.5

1.0

1.5

0.5

CONTROL TEST

1.0

1.5

0.5

CONTROL TEST

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

♂ ♀AAB

BBA

BAA

ABA

AAB

ABA

AAB

AAB

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 1

Family 2Preimplantation genetic diagnosis

for cystic fibrosis

Whole genome amplification by isothermal multiple displacementamplification of 2-10 cells in eachbiopsy/embryo

Embryo 7

13, 16, 18, 21, 22Monosomy 18, 21, 22

X, Y, 21Trisomy XMonosomy 21

Array CGH

Karyomap

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