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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)

n Hormonal stimulation of the ovaries to
  produce mature oocytes.
n Intracytoplasmic sperm injection (ICSI) or
  in vitro fertilisation (IVF).
n Embryo Biopsy
n Genetic analysis of one or two cells
   - PCR or FISH.
       Embryo Development following
IVF        fertilisation (day 0-2)




ICSI      Fertilised egg   2 cell embryo   4 cell embryo
Cleavage stage biopsy
               Project Aims
n   Whole genome amplification: small cell
    numbers
    - Buccal cells: 1 / 2 /5 / multiple cells
    - (Blastomeres:1 / 2 /5 / multiple cells ?)
n   Theoretical microsatellite marker evaluation,
    validation & incorporation into multiplex assays.
n   Marker segregation analysis.
n   Application of multiplex assays to WGA
    products.
Schematic of buccal cell collection,
        rinsing and lysis
                           Small group of
                           nucleated cells are
                           transferred from the
                           cell suspension
                       A
                   1

                   2


      suspension
 cell suspension   3   B


                             Media from pipette is
                             emptied into here after
                             each transfer.
Whole Genome Amplification (WGA)

n Produces large quantities of DNA from
  small templates.
n Overcomes problems with single cell
  lysates.
n 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

n Amplification products assessed using
  QF-PCR assay for the detection of common
  aneuploidies.
n 12 tetra nucleotide repeat markers for
  chromosomes 13, 18 and 21.
n 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

n Allele drop out (ADO) more disruptive
  to direct mutation test:
- False positives
- False negatives
n­ number of markers, ­  chances of a
  result.
Preimplantation Genetic Haplotyping (PGH)

n   Applicable to any single gene disorder in which
    the causative gene has been mapped.
n   Microsatellite markers span disease locus.
n   Multiplex assays create dense haplotypes
n   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)

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

D7S523     D7S2554     D7S486 IVS1CA Phe508 CFSTR1              D7S643 D7S650   D72490
5.4Mb       2.7 Mb      1.2 Mb   69.4 Kb      0.3 Mb             3.6 Mb 3.7Mb   5.5Mb

  



                                      CFTR




                 D7S2502 D7S2460 IVS8CA           D7S2847            D7S480
                     1.7Mb   0.7Mb   11.3Kb            1.5 Mb         3.7Mb
         Removal of two least useful markers


D7S523     D7S2554     D7S486 IVS1CA Phe508 CFSTR1              D7S643 D7S650   D72490
5.4Mb       2.7 Mb      1.2 Mb   69.4 Kb      0.3 Mb             3.6 Mb 3.7Mb   5.5Mb

  



                                      CFTR




                 D7S2502 D7S2460 IVS8CA           D7S2847            D7S480
                     1.7Mb   0.7Mb   11.3Kb            1.5 Mb         3.7Mb
       Selection and evaluation of
    theoretical polymorphic markers

n   20 microsatellite markers selected.
n   Primer selection using Primer 3.
n   Markers evaluated individually.
n   Incorporate markers into assay.
n   Calculate Polymorphism Information Content
    (PIC) & Heterozygosity (HET) scores.
          PIC & HET values

 Marker     HET Score   PIC Score
   MS1         0.87       0.86
   MS3         0.90       0.89
   MS6         0.76       0.72
   MS7         0.53       0.51
  MS15         0.68       0.64
  MS19         0.86       0.84

(n=192)
      Addition of new markers to two tube PGH
            assay for Cystic Fibrosis (CF)

    MS1                       MS3                           MS6
    4.6Mb                     0.7 Mb                       2.6 Mb

            D7S2554     D7S486 IVS1CA Phe508 CFSTR1                 D7S643 D7S650
             2.7 Mb      1.2 Mb     69.4 Kb      0.3 Mb              3.6 Mb 3.7Mb



                                         CFTR




                  D7S2502 D7S2460 IVS8CA                D7S2847          D7S480
                      1.7Mb   0.7Mb    11.3Kb             1.5 Mb          3.7Mb
                                                MS1
                                                9
                                                0.4Mb
Multiplex A
Multiplex B
Typical CF haplotypes from family
            analysis
Buccal cells vs lymphocytes
        WGA of blastomeres

n Discarded embryos.
n Embryo’s biopsied.
n Single cell removed and lysed.
n Remainder of embryo lysed (used as
 comparison).
         Preliminary embryo data
1/10                           1/6




9/10                            5/6
  9/10




1/6                           1/10




5/6                           9/10
                  Conclusions
n    WGA from small cell numbers was successful.
n   Four new polymorphic markers found close to CFTR.
n   Markers incorporated into CF assay.
n    Highly informative haplotypes –universally
    applicable.
n   Assay suitable for WGA DNA from small cell
    numbers.
n   Preliminary embryo data is promising!
           Acknowledgements
n   Pamela Renwick, Jane Trussler & Cheryl Black
    (Guys’ and St Thomas’Hospital, London).
n   Sue Pickering (Assisted Conception Unit,
    Edinburgh).
n   Jon Warner & Paul Westwood (Edinburgh
    Molecular Genetics).
n   Everyone in the Edinburgh Molecular Genetics
    Lab.

				
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