Evaluation of whole genome amplification from small cell numbers

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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.

 Intracytoplasmic sperm injection (ICSI) or in vitro fertilisation (IVF).

 Embryo Biopsy

 Genetic analysis of one or two cells

- PCR or FISH.

IVF

Embryo Development following fertilisation (day 0-2)

ICSI

Fertilised egg 2 cell embryo 4 cell embryo

Cleavage stage biopsy

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

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

Whole Genome Amplification:

Multiple Displacement Amplification

(MDA)

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

MDA products electrophoresed on a 2% agarose gel

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 10 11 12 13 14 15 16 L

L 1 2 3 4 5 6 7 8 9 10 11 12 L

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.

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

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

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

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

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)

 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)

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.

D7S523

5.4Mb

Guys’ and St Thomas’ two tube PGH assay for Cystic Fibrosis (CF)

D7S2554

2.7 Mb

D7S486

1.2 Mb

IVS1CA

69.4 Kb

Phe508 CFSTR1

0.3 Mb

D7S643

3.6 Mb

D7S650

3.7Mb

D72490

5.5Mb

D7S2502

1.7Mb

D7S2460

0.7Mb

IVS8CA

11.3Kb

D7S2847

1.5 Mb

D7S480

3.7Mb

D7S523

5.4Mb

Removal of two least useful markers

D7S2554

2.7 Mb

D7S486

1.2 Mb

IVS1CA

69.4 Kb

Phe508 CFSTR1

0.3 Mb

D7S643

3.6 Mb

D7S650

3.7Mb

D72490

5.5Mb

D7S2502

1.7Mb

D7S2460

0.7Mb

IVS8CA

11.3Kb

D7S2847

1.5 Mb

D7S480

3.7Mb

Selection and evaluation of theoretical polymorphic markers

1.

2.

3.

4.

5.

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

Marker HET Score PIC Score

MS1 0.87

0.86

MS3

MS6

MS7

MS15

MS19

0.90

0.76

0.53

0.68

0.86

0.89

0.72

0.51

0.64

0.84

(n=192)

Addition of new markers to two tube PGH assay for Cystic Fibrosis (CF)

MS1

4.6Mb

D7S2554

2.7 Mb

MS3

0.7 Mb

D7S486

1.2 Mb

IVS1CA

69.4 Kb

Phe508 CFSTR1

0.3 Mb

MS6

2.6 Mb

D7S643

3.6 Mb

D7S650

3.7Mb

D7S2502

1.7Mb

D7S2460

0.7Mb

IVS8CA

11.3Kb

MS19

0.4Mb

D7S2847

1.5 Mb

D7S480

3.7Mb

Multiplex A

Multiplex B

Typical CF haplotypes from family analysis

Buccal cells vs lymphocytes

WGA of blastomeres

 Discarded embryos.

Embryo’s biopsied.

 Single cell removed and lysed.

 Remainder of embryo lysed (used as comparison).

1/10

Preliminary embryo data

1/6

5/6

1/6 1/10

5/6 9/10

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

 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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