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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 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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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

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.


Embryo development following fertilisation day 0 2

Embryo Development following fertilisation (day 0-2)

IVF

ICSI

Fertilised egg

2 cell embryo

4 cell embryo


Jenna mcluskey edinburgh molecular genetics service

Cleavage stage biopsy


Project aims

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.


Schematic of buccal cell collection rinsing and lysis

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


Whole genome amplification wga

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


Jenna mcluskey edinburgh molecular genetics service

Whole Genome Amplification:Multiple Displacement Amplification (MDA)

A. Mamone, 2003, Amersham Biosciences, Piscataway, NJ, USA


Mda products electrophoresed on a 2 agarose gel

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


Validation of wga dna products

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.


Jenna mcluskey edinburgh molecular genetics service

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


Jenna mcluskey edinburgh molecular genetics service

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


Jenna mcluskey edinburgh molecular genetics service

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


Jenna mcluskey edinburgh molecular genetics service

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


Direct mutation testing vs haplotyping

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.


Preimplantation genetic haplotyping pgh

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


Guys and st thomas two tube pgh assay for cystic fibrosis cf

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.


Guys and st thomas two tube pgh assay for cystic fibrosis cf1

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)


Removal of two least useful markers

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


Selection and evaluation of theoretical polymorphic markers

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.


Pic het values

PIC & HET values

(n=192)


Addition of new markers to two tube pgh assay for cystic fibrosis cf

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


Multiplex a

Multiplex A


Multiplex b

Multiplex B


Typical cf haplotypes from family analysis

Typical CF haplotypes from family analysis


Buccal cells vs lymphocytes

Buccal cells vs lymphocytes


Wga of blastomeres

WGA of blastomeres

  • Discarded embryos.

  • Embryo’s biopsied.

  • Single cell removed and lysed.

  • Remainder of embryo lysed (used as comparison).


Preliminary embryo data

Preliminary embryo data

1/10

1/6

5/6

9/10

9/10

1/6

1/10

5/6

9/10


Conclusions

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!


Acknowledgements

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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