Gabbay Award Lecture

1,863 views
1,703 views

Published on

This is the Gabbay Award lecture I presented at Brandeis University in Boston on Nov 16th 2009

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
1,863
On SlideShare
0
From Embeds
0
Number of Embeds
16
Actions
Shares
0
Downloads
36
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Gabbay Award Lecture

  1. 1. ‘ Designer babies’: twenty years on Alan H Handyside London Bridge Fertility, Gynaecology And Genetics Centre London United Kingdom Gabbay Award Lecture Brandeis University November 16 th , 2009
  2. 3. <ul><li>In Vitro Fertilisation (IVF) </li></ul><ul><ul><ul><li>Downregulation of ovarian folliculogenesis </li></ul></ul></ul><ul><ul><ul><li>Superovulation and egg collection </li></ul></ul></ul><ul><ul><ul><li>Intracytoplasmic sperm microinjection (ICSI) and embryo culture </li></ul></ul></ul><ul><ul><ul><li>Cleavage stage biopsy by zona drilling and micromanipulation </li></ul></ul></ul><ul><li>Single cell genetic analysis (12-72h) </li></ul><ul><li>Selective transfer of unaffected embryos 3-5 days post fertilisation </li></ul>Preimplantation Genetic Diagnosis (PGD)
  3. 4. 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
  4. 5. Inherited conditions diagnosed by PGD <ul><li>Familial polyposis coli </li></ul><ul><li>Retinoblastoma </li></ul><ul><li>Li-Fraumeni syndrome </li></ul><ul><li>Chromosomal abnormalities </li></ul><ul><li>Aneuploidy </li></ul><ul><li>Translocations </li></ul><ul><li>Structural </li></ul><ul><li>Other </li></ul><ul><li>HLA matching </li></ul>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
  5. 6. Strategies for single cell genetic analysis SINGLE OR MULTIPLE CELL BIOPSY LYSIS NESTED PCR OUTER PCR MULTIPLEX F-PCR MULTIPLEX PCR PEP/DOP-PCR INNER PCR HETERODUPLEX FORMATION PAGE MDA AUTOMATED SEQUENCER SEQUENCING MINISEQUENCING AUTOMATED SEQUENCER SEQUENCING MINISEQUENCING AUTOMATED SEQUENCER MULTIPLEX PCR ANY COMBINATION OF CONVENTIONAL DNA BASED TESTS CGH MICROARRAYS A B C D E CGH DNA free reagents and conditions
  6. 7. Fiorentino et al (2005) Eur J Hum Genet 13, 953
  7. 8. Handyside et al (2004) Mol Hum Reprod 10, 167
  8. 9. Heterozygous Allele dropout Amp failure Allele specific preferential amplification 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 1 2
  9. 10. Preimplantation genetic haplotyping following MDA from single cells Renwick et al (2006) Reprod Biomed Online 13, 110
  10. 11. Embryo 2 Monosomy 16, 21 Trisomy 22
  11. 12. Chromosome imbalance and genotyping by microarrays <ul><li>DNA microarrays with probes spaced typically at 1 Mb intervals genome wide for comparative genomic hybridisation (array CGH) </li></ul><ul><li>High density genome wide single nucleotide polymorphism (SNP) genotyping arrays </li></ul>
  12. 13. CONTROL TEST
  13. 14. CONTROL TEST 1.0 1.5 0.5
  14. 15. CONTROL TEST 1.0 1.5 0.5
  15. 16. CONTROL TEST 1.0 1.5 0.5
  16. 17. First or first and second polar body biopsy on day 0 or day 1 Array comparative genomic hybridisation (CGH) following whole genome amplification
  17. 18. <ul><li>Single nucleotide polymorphisms (SNPs) </li></ul><ul><li>10 million SNPs across human genome </li></ul><ul><li>Many biallelic (AA, AB, BB) </li></ul><ul><li>Major contribution to genetic diversity, inherited disease and variants associated with common multifactorial conditions </li></ul>
  18. 19. Karyomapping <ul><li>A universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes </li></ul><ul><li>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) </li></ul><ul><li>Mendelian analysis and karyomapping of the parental and grandparental haplotypes for each chromosome or chromosome segment in recombinant chromosomes </li></ul>Handyside et al (2009) J Med Genet Online First
  19. 20. ♂ ♀ A A B B B A B A A A B A A A B A B A A A B A A B
  20. 21. 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).
  21. 22. Family 1
  22. 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
  23. 24. Embryo 7 13, 16, 18, 21, 22 Monosomy 18, 21, 22 X, Y, 21 Trisomy X Monosomy 21 Array CGH Karyomap
  24. 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   
  25. 26. <ul><li>A universal method for genome wide analysis of genetic defects and their parental origin </li></ul><ul><li>Complementary to standard methods of SNP data analysis for molecular cytogenetics </li></ul><ul><li>Parental origin of aneuploidies and meiotic phase of origin of trisomies identified </li></ul><ul><li>Inheritance and parental origin of copy number variants and structural chromosomal abnormalities can be analysed </li></ul><ul><li>Enhanced linkage based analysis of loci genome wide for diagnosis of single gene defects and analysis of recurrence risk of multifactorial conditions </li></ul><ul><li>Applicable at the single cell level for preimplantation genetic diagnosis (PGD) </li></ul>SNP analysis and Karyomapping
  26. 27. <ul><li>Genoma Laboratory, Rome </li></ul><ul><ul><li>Anil Biricik </li></ul></ul><ul><ul><li>Francesco Fiorentino </li></ul></ul><ul><li>Bridge Genoma, London </li></ul><ul><ul><li>Helen Tempest </li></ul></ul><ul><ul><li>Mira Grigorova </li></ul></ul><ul><ul><li>Alan Thornhill </li></ul></ul><ul><li>Department of Biosciences, University of Kent </li></ul><ul><ul><li>Alem Gebrasselasi </li></ul></ul><ul><ul><li>Darren Griffin </li></ul></ul><ul><li>BlueGnome, Cambridge </li></ul><ul><ul><li>Anthony Brown </li></ul></ul><ul><ul><li>Tony Gordon </li></ul></ul><ul><ul><li>Graham Snudden </li></ul></ul><ul><ul><li>Nick Haan </li></ul></ul><ul><li>Department of Pathology, University of Cambridge </li></ul><ul><ul><li>Ben Dunmore </li></ul></ul><ul><ul><li>Nabeel Affara </li></ul></ul><ul><li>Genetics and IVF Institute, </li></ul><ul><li>Fairfax, Virginia </li></ul><ul><ul><li>Gary Harton </li></ul></ul><ul><ul><li>Brian Mariani </li></ul></ul>Acknowledgements

×