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

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Presentation on theme: "Evaluation of whole genome amplification from small cell numbers and the development of pre-implantation genetic haplotyping assays Jenna McLuskey Edinburgh."— Presentation transcript:

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 Edinburgh Molecular Genetics Service

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

3 Embryo Development following fertilisation (day 0-2) IVF ICSI Fertilised egg2 cell embryo4 cell embryo

4 Cleavage stage biopsy

5 Project Aims Whole genome amplification: small cell numbers 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. Theoretical microsatellite marker evaluation, validation & incorporation into multiplex assays. Marker segregation analysis. Marker segregation analysis. Application of multiplex assays to WGA products. Application of multiplex assays to WGA products.

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

7 Whole Genome Amplification (WGA) Produces large quantities of DNA from small templates. Produces large quantities of DNA from small templates. Overcomes problems with single cell lysates. Overcomes problems with single cell lysates. Successful PCR amplification, following WGA for 5/5 single lymphocytes and 10/11 single blastomeres. 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 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 3 4 5 6 7 8 910 11 12 13 14 15 L L 16 L 1 2 3 4 5 6 7 8 9 10 L L 1 2 3 4 5 6 7 8 9 L L 1 2 3 4 5 6 7 8 9 L 12 11 2 MDA products electrophoresed on a 2% agarose gel

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

11 IFNAR D211411 blood lymphocytes five buccal cells D21S1437 D21S11 D13S628D13S634D18S535 blood lymphocytes five buccal cells blood lymphocytes five buccal cells D18S1002 D18S391 D13S742D18S386 D13S305

12 IFNAR D211411 blood lymphocytes five buccal cells D21S1437 D21S11 D13S628D13S634D18S535 blood lymphocytes five buccal cells blood lymphocytes five buccal cells D18S1002 D18S391 D13S742 D18S386 D13S305

13 IFNAR D211411 blood lymphocytes five buccal cells D21S1437 D21S11 D13S628D13S634D18S535 blood lymphocytes five buccal cells blood lymphocytes five buccal cells D18S1002 D18S391 D13S742 D18S386 D13S305

14 IFNAR D211411 blood lymphocytes five buccal cells D21S1437 D21S11 D13S628D13S634D18S535 blood lymphocytes five buccal cells blood lymphocytes five buccal cells D18S1002 D18S391 D13S742 D18S386 D13S305

15 Direct mutation testing vs haplotyping Allele drop out (ADO) more disruptive to direct mutation test: Allele drop out (ADO) more disruptive to direct mutation test: - False positives - False positives - False negatives - False negatives  number of markers,  chances of a result.  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. Applicable to any single gene disorder in which the causative gene has been mapped. Microsatellite markers span disease locus. Microsatellite markers span disease locus. Multiplex assays create dense haplotypes 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 – 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 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 PGH assay for Cystic Fibrosis (CF) PGH for CF currently offered at Guys’ and St Thomas’ Hospital, London. PGH for CF currently offered at Guys’ and St Thomas’ Hospital, London. Two tube universal tagged fluorescent multiplex. Two tube universal tagged fluorescent multiplex. Ten dinucleotide & 3 tetranucleotide repeat markers spanning the CFTR locus. Ten dinucleotide & 3 tetranucleotide repeat markers spanning the CFTR locus.

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

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

20 Selection and evaluation of theoretical polymorphic markers 1. 20 microsatellite markers selected. 2. Primer selection using Primer 3. 3. Markers evaluated individually. 4. Incorporate markers into assay. 5. Calculate Polymorphism Information Content (PIC) & Heterozygosity (HET) scores.

21

22 PIC & HET values Marker HET Score PIC Score MS10.870.86 MS30.900.89 MS60.760.72 MS70.530.51 MS150.680.64 MS190.860.84 (n=192)

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

24 Multiplex A

25 Multiplex B

26 Typical CF haplotypes from family analysis

27 Buccal cells vs lymphocytes

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

29 Preliminary embryo data 1/10 9/10 1/6 5/6 1/6 5/6 1/10 9/10

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

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

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