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

Evaluation of whole genome amplification from small cell numbers

and the development of pre-implantation genetic haplotyping assays

Jenna McLuskeyJenna McLuskey

Edinburgh Molecular Genetics ServiceEdinburgh Molecular Genetics Service

Preimplantation Genetic Diagnosis (PGD)

Hormonal stimulation of the ovaries to Hormonal stimulation of the ovaries to produce mature oocytes.produce mature oocytes.

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

Embryo BiopsyEmbryo Biopsy Genetic analysis of one or two cellsGenetic analysis of one or two cells

- PCR or FISH.- PCR or FISH.

Embryo Development following fertilisation (day 0-2)IVF

ICSI Fertilised egg 2 cell embryo 4 cell embryo

Cleavage stage biopsy

Project Aims

Whole genome amplification: small cell Whole genome amplification: small cell numbersnumbers- Buccal cells:- Buccal cells: 1 / 2 /5 / multiple cells1 / 2 /5 / multiple cells- (Blastomeres:1 / 2 /5 / multiple cells ?)- (Blastomeres:1 / 2 /5 / multiple cells ?)

Theoretical microsatellite marker evaluation, Theoretical microsatellite marker evaluation, validation & incorporation into multiplex assays.validation & incorporation into multiplex assays.

Marker segregation analysis.Marker segregation analysis. Application of multiplex assays to WGA Application of multiplex assays to WGA

products.products.

Schematic of buccal cell collection,

rinsing and lysis

1

2

3

A

B

1

2

3

A

Bcell 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

Bcell suspension

Small group of nucleated cells are transferred from the cell suspension

Media from pipette is emptied into here after each transfer.

Whole Genome Amplification (WGA)

Produces large quantities of DNA from Produces large quantities of DNA from small templates.small templates.

Overcomes problems with single cell Overcomes problems with single cell lysates.lysates.

Successful PCR amplification, following Successful PCR amplification, following WGA for 5/5 single lymphocytes and 10/11 WGA for 5/5 single lymphocytes and 10/11 single blastomeressingle blastomeres..

Handyside et al (2004) Isothermal whole genome amplification from single and small 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; numbers of cells: a new era for PGD of inherited diseases. Molecular Reproduction 10; 767-772767-772

Whole Genome Amplification:Multiple Displacement Amplification

(MDA)

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

1 3 4 5 6 7 8 9 10 11 12 13 14 15L

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 L10 12112

MDA products electrophoresed on a 2% agarose gel

Validation of WGA DNA products

Amplification products assessed using Amplification products assessed using

QF-PCR assay for the detection of common QF-PCR assay for the detection of common aneuploidies.aneuploidies.

12 tetra nucleotide repeat markers for 12 tetra nucleotide repeat markers for chromosomes 13, 18 and 21.chromosomes 13, 18 and 21.

PCR products amplified from WGA DNA PCR products amplified from WGA DNA vs DNA extracted from blood lymphocytes.vs DNA extracted from blood lymphocytes.

IFNAR D211411

blood lymphocytes

five buccal cells

D21S1437 D21S11 D13S628 D13S634 D18S535

blood lymphocytes

five buccal cells

blood lymphocytes

five buccal cellsD18S1002 D18S391 D13S742 D18S386 D13S305

IFNAR D211411

blood lymphocytes

five buccal cells

D21S1437 D21S11 D13S628 D13S634 D18S535

blood lymphocytes

five buccal cells

blood lymphocytes

five buccal cellsD18S1002 D18S391 D13S742 D18S386 D13S305

IFNAR D211411

blood lymphocytes

five buccal cells

D21S1437 D21S11 D13S628 D13S634 D18S535

blood lymphocytes

five buccal cells

blood lymphocytes

five buccal cellsD18S1002 D18S391 D13S742 D18S386 D13S305

IFNAR D211411

blood lymphocytes

five buccal cells

D21S1437 D21S11 D13S628 D13S634 D18S535

blood lymphocytes

five buccal cells

blood lymphocytes

five buccal cellsD18S1002 D18S391 D13S742 D18S386 D13S305

Direct mutation testing vs haplotyping

Allele drop out (ADO) more disruptive Allele drop out (ADO) more disruptive to direct mutation test:to direct mutation test:

- False positives- False positives

- False negatives- False negatives number of markers, number of markers, chances of a chances of a

result.result.

Preimplantation Genetic Haplotyping (PGH)

Applicable to any single gene disorder in which Applicable to any single gene disorder in which the causative gene has been mapped.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 Renwick et al – 12 closely linked microsatellite

markers – 93% haplotypes constructed despite markers – 93% haplotypes constructed despite some ADO at individual loci.some ADO at individual loci.

Renwick et al (2006) Proof of Principle and first cases using PGH – a paradigm shift for Renwick et al (2006) Proof of Principle and first cases using PGH – a paradigm shift for

embryo diagnosis. Reproductive Medicine 13; 758-767embryo diagnosis. Reproductive Medicine 13; 758-767

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

PGH for CF currently offered at Guys’ and PGH for CF currently offered at Guys’ and St Thomas’ Hospital, London.St Thomas’ Hospital, London.

Two tube universal tagged fluorescent Two tube universal tagged fluorescent multiplex.multiplex.

Ten dinucleotide & 3 tetranucleotide repeat Ten dinucleotide & 3 tetranucleotide repeat markers spanning the markers spanning the CFTR CFTR locus.locus.

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

D724905.5Mb

D7S5235.4Mb

CFTR

IVS8CA

IVS1CA

D7S2847

D7S643

D7S480

D7S650

D7S2460D7S2502

D7S2554 Phe508

11.3Kb

69.4 KbCFSTR10.3 Mb

1.5 Mb

3.6 Mb

3.7Mb

3.7Mb

0.7Mb

D7S4861.2 Mb

1.7Mb

2.7 Mb

CFTR

Removal of two least useful markers

D724905.5Mb

D7S5235.4Mb

CFTR

IVS8CA

IVS1CA

D7S2847

D7S643

D7S480

D7S650

D7S2460D7S2502

D7S2554 Phe508

11.3Kb

69.4 KbCFSTR10.3 Mb

1.5 Mb

3.6 Mb

3.7Mb

3.7Mb

0.7Mb

D7S4861.2 Mb

1.7Mb

2.7 Mb

CFTR

Selection and evaluation of theoretical polymorphic markers

1.1. 20 microsatellite markers selected.20 microsatellite markers selected.

2.2. Primer selection using Primer 3.Primer selection using Primer 3.

3.3. Markers evaluated individually.Markers evaluated individually.

4.4. Incorporate markers into assay.Incorporate markers into assay.

5.5. Calculate Polymorphism Information Content Calculate Polymorphism Information Content (PIC) & Heterozygosity (HET) scores.(PIC) & Heterozygosity (HET) scores.

PIC & HET values

MarkerMarker HET ScoreHET Score PIC ScorePIC Score

MS1MS1 0.870.87 0.860.86

MS3MS3 0.900.90 0.890.89

MS6MS6 0.760.76 0.720.72

MS7MS7 0.530.53 0.510.51

MS15MS15 0.680.68 0.640.64

MS19MS19 0.860.86 0.840.84

(n=192)

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

MS3MS14.6Mb 0.7 Mb 2.6 Mb

0.4Mb

MS6

MS19

CFTR

IVS8CA

IVS1CA

D7S2847

D7S643

D7S480

D7S650

D7S2460D7S2502

D7S2554 Phe508

11.3Kb

69.4 KbCFSTR10.3 Mb

1.5 Mb

3.6 Mb

3.7Mb

3.7Mb

0.7Mb

D7S4861.2 Mb

1.7Mb

2.7 Mb

CFTR

Multiplex A

Multiplex B

Typical CF haplotypes from family analysis

247181331227255289332226264252190292252291289

243187335228245289334240256260183288252302304

243181333227243290331228268258194290243289306

229183333229245289331228260256194275250302289

229183333229245289331228260256194275250302289

243187335228245289334240256260183288252302304

243181333227243290331228268258194290243289306

247181331227255289332226264252190292252291289

MS1D7S2554D7S2502D7S486MS3D7S2460IVS1CAIVS8CACFSTR1MS19D7S2847MS6D7S643D7S480D7S650

MARKER LOCATION KEY

247181331227255289332226264252190292252291289

243187335228245289334240256260183288252302304

243181333227243290331228268258194290243289306

229183333229245289331228260256194275250302289

229183333229245289331228260256194275250302289

243187335228245289334240256260183288252302304

243181333227243290331228268258194290243289306

247181331227255289332226264252190292252291289

MS1D7S2554D7S2502D7S486MS3D7S2460IVS1CAIVS8CACFSTR1MS19D7S2847MS6D7S643D7S480D7S650

MARKER LOCATION KEY

MS1D7S2554D7S2502D7S486MS3D7S2460IVS1CAIVS8CACFSTR1MS19D7S2847MS6D7S643D7S480D7S650

MS1D7S2554D7S2502D7S486MS3D7S2460IVS1CAIVS8CACFSTR1MS19D7S2847MS6D7S643D7S480D7S650

MARKER LOCATION KEY

Lymphocytes

Buccal cells

Lymphocytes

Buccal cells

Lymphocytes

Buccal cells

Lymphocytes

Buccal cells

Lymphocytes

Buccal cells

Lymphocytes

Buccal cells

Buccal cells vs lymphocytes

Lymphocytes

Buccal cells

Lymphocytes

Buccal cells

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 Remainder of embryo lysed (used as

comparison).comparison).

Preliminary embryo data

1/10

9/109/10

1/6

5/6

1/6

5/6

1/10

9/10

Conclusions

WGA from small cell numbers was successful.WGA from small cell numbers was successful. Four new polymorphic markers found close to Four new polymorphic markers found close to CFTRCFTR.. Markers incorporated into CF assay.Markers incorporated into CF assay. Highly informative haplotypes –universally Highly informative haplotypes –universally

applicable.applicable. Assay suitable for WGA DNA from small cell Assay suitable for WGA DNA from small cell

numbers.numbers. Preliminary embryo data is promising!Preliminary embryo data is promising!

Acknowledgements

Pamela Renwick, Jane Trussler & Cheryl Black Pamela Renwick, Jane Trussler & Cheryl Black (Guys’ and St Thomas’Hospital, London).(Guys’ and St Thomas’Hospital, London).

Sue Pickering (Assisted Conception Unit, Sue Pickering (Assisted Conception Unit, Edinburgh).Edinburgh).

Jon Warner & Paul Westwood (Edinburgh Jon Warner & Paul Westwood (Edinburgh Molecular Genetics).Molecular Genetics).

Everyone in the Edinburgh Molecular Genetics Everyone in the Edinburgh Molecular Genetics Lab.Lab.