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Jenna McLuskey Edinburgh Molecular Genetics Service

Evaluation of whole genome amplification from small cell numbers and the development of pre-implantation genetic haplotyping assays. Jenna McLuskey Edinburgh Molecular Genetics Service. Preimplantation Genetic Diagnosis (PGD). Hormonal stimulation of the ovaries to produce mature oocytes.

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Jenna McLuskey Edinburgh Molecular Genetics Service

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  1. Evaluation of whole genome amplification from small cell numbers and the development of pre-implantation genetic haplotyping assays Jenna McLuskey Edinburgh Molecular Genetics Service

  2. Preimplantation Genetic Diagnosis (PGD) • Hormonal stimulation of the ovaries to produce mature oocytes. • Intracytoplasmic sperm injection (ICSI) or in vitro fertilisation (IVF). • Embryo Biopsy • Genetic analysis of one or two cells - PCR or FISH.

  3. Embryo Development following fertilisation (day 0-2) IVF ICSI Fertilised egg 2 cell embryo 4 cell embryo

  4. Cleavage stage biopsy

  5. Project Aims • Whole genome amplification: small cell numbers - Buccal cells: 1 / 2 /5 / multiple cells - (Blastomeres:1 / 2 /5 / multiple cells ?) • Theoretical microsatellite marker evaluation, validation & incorporation into multiplex assays. • Marker segregation analysis. • Application of multiplex assays to WGA products.

  6. Small group of Small group of nucleated cells are nucleated cells are transferred from the transferred from the cell suspension cell suspension A A A A 1 1 1 1 2 2 2 2 3 3 3 3 B B B B cell suspension cell suspension Media from pipette is Media from pipette is emptied into here after emptied into here after each transfer. each transfer. Schematic of buccal cell collection, rinsing and lysis

  7. Whole Genome Amplification (WGA) • Produces large quantities of DNA from small templates. • Overcomes problems with single cell lysates. • Successful PCR amplification, following WGA for 5/5 single lymphocytes and 10/11 single blastomeres. Handyside et al (2004) Isothermal whole genome amplification from single and small numbers of cells: a new era for PGD of inherited diseases. Molecular Reproduction 10; 767-772

  8. Whole Genome Amplification:Multiple Displacement Amplification (MDA) A. Mamone, 2003, Amersham Biosciences, Piscataway, NJ, USA

  9. 1 4 L 3 5 2 8 6 7 9 2 10 1 L 3 5 L 4 6 7 9 8 L 1 4 L 3 5 2 4 9 3 6 2 5 7 8 6 10 7 L 1 L 11 11 14 12 13 9 10 12 8 15 16 L MDA products electrophoresed on a 2% agarose gel

  10. Validation of WGA DNA products • Amplification products assessed using QF-PCR assay for the detection of common aneuploidies. • 12 tetra nucleotide repeat markers for chromosomes 13, 18 and 21. • PCR products amplified from WGA DNA vs DNA extracted from blood lymphocytes.

  11. blood lymphocytes five buccal cells D21S1437 D21S11 D13S628 D13S634 D18S535 blood lymphocytes five buccal cells D18S1002 D18S391 D13S742 D18S386 D13S305 blood lymphocytes five buccal cells IFNAR D211411

  12. blood lymphocytes five buccal cells D21S1437 D21S11 D13S628 D13S634 D18S535 blood lymphocytes five buccal cells D18S1002 D18S391 D13S742 D13S305 D18S386 blood lymphocytes five buccal cells IFNAR D211411

  13. blood lymphocytes five buccal cells D21S1437 D21S11 D13S628 D13S634 D18S535 blood lymphocytes five buccal cells D18S1002 D18S391 D13S742 D13S305 D18S386 blood lymphocytes five buccal cells IFNAR D211411

  14. blood lymphocytes five buccal cells D21S1437 D21S11 D13S628 D13S634 D18S535 blood lymphocytes five buccal cells D18S1002 D18S391 D13S742 D13S305 D18S386 blood lymphocytes five buccal cells IFNAR D211411

  15. Direct mutation testing vs haplotyping • Allele drop out (ADO) more disruptive to direct mutation test: - False positives - False negatives •  number of markers,  chances of a result.

  16. Preimplantation Genetic Haplotyping (PGH) • Applicable to any single gene disorder in which the causative gene has been mapped. • Microsatellite markers span disease locus. • Multiplex assays create dense haplotypes • Renwick et al – 12 closely linked microsatellite markers – 93% haplotypes constructed despite some ADO at individual loci. Renwick et al (2006) Proof of Principle and first cases using PGH – a paradigm shift for embryo diagnosis. Reproductive Medicine 13; 758-767

  17. Guys’ and St Thomas’ two tube PGHassay for Cystic Fibrosis (CF) • PGH for CF currently offered at Guys’ and St Thomas’ Hospital, London. • Two tube universal tagged fluorescent multiplex. • Ten dinucleotide & 3 tetranucleotide repeat markers spanning the CFTR locus.

  18. D72490 IVS1CA Phe508 CFSTR1 D7S650 D7S643 D7S486 D7S2554 D7S523 0.3 Mb 5.5Mb 3.6 Mb 69.4 Kb 1.2 Mb 3.7Mb 2.7 Mb 5.4Mb CFTR CFTR D7S2502 IVS8CA D7S2460 D7S2847 D7S480 1.7Mb 0.7Mb 3.7Mb 11.3Kb 1.5 Mb Guys’ and St Thomas’ two tube PGHassay for Cystic Fibrosis (CF)

  19. D72490 IVS1CA Phe508 CFSTR1 D7S650 D7S643 D7S486 D7S2554 D7S523 0.3 Mb 5.5Mb 3.6 Mb 69.4 Kb 1.2 Mb 3.7Mb 2.7 Mb 5.4Mb CFTR CFTR D7S2502 IVS8CA D7S2460 D7S2847 D7S480 1.7Mb 0.7Mb 3.7Mb 11.3Kb 1.5 Mb Removal of two least useful markers

  20. Selection and evaluation of theoretical polymorphic markers • 20 microsatellite markers selected. • Primer selection using Primer 3. • Markers evaluated individually. • Incorporate markers into assay. • Calculate Polymorphism Information Content (PIC) & Heterozygosity (HET) scores.

  21. PIC & HET values (n=192)

  22. Addition of new markers to two tube PGHassay for Cystic Fibrosis (CF) MS1 MS3 MS6 2.6 Mb 0.7 Mb 4.6Mb IVS1CA Phe508 CFSTR1 D7S650 D7S643 D7S486 D7S2554 0.3 Mb 3.6 Mb 69.4 Kb 1.2 Mb 3.7Mb 2.7 Mb CFTR CFTR D7S2502 IVS8CA D7S2460 D7S2847 D7S480 1.7Mb 0.7Mb 3.7Mb 11.3Kb 1.5 Mb MS19 0.4Mb

  23. Multiplex A

  24. Multiplex B

  25. Typical CF haplotypes from family analysis

  26. Buccal cells vs lymphocytes

  27. WGA of blastomeres • Discarded embryos. • Embryo’s biopsied. • Single cell removed and lysed. • Remainder of embryo lysed (used as comparison).

  28. Preliminary embryo data 1/10 1/6 5/6 9/10 9/10 1/6 1/10 5/6 9/10

  29. Conclusions • WGA from small cell numbers was successful. • Four new polymorphic markers found close to CFTR. • Markers incorporated into CF assay. • Highly informative haplotypes –universally applicable. • Assay suitable for WGA DNA from small cell numbers. • Preliminary embryo data is promising!

  30. Acknowledgements • Pamela Renwick, Jane Trussler & Cheryl Black (Guys’ and St Thomas’Hospital, London). • Sue Pickering (Assisted Conception Unit, Edinburgh). • Jon Warner & Paul Westwood (Edinburgh Molecular Genetics). • Everyone in the Edinburgh Molecular Genetics Lab.

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