1. PGD for single gene disorders
An update from Edinburgh
Sally Morton, Genetic Counsellor
23 November 2010

2. Topics covered Principle of PGD and haplotyping
Criteria to receive treatment in Edinburgh
Pilot study update
What should couples expect?
Which family samples required?

3. Human preimplantation
Development day 0-2
IVF
ICSI
Fertilised egg
2 cell embryo
4 cell embryo
Stimulatory drugs to create several eggs
Fertilised by ICSI to reduce risk of contamination

4. Cleavage stage Biopsy

6. Placement of CF markers with respect to p.Phe508del
MS1
MS3
MS6
4.6Mb
0.7 Mb
2.6 Mb
D7S2554
D7S486
IVS1CA
CFSTR1
D7S643
D7S650
2.7 Mb
1.2 Mb
69.4 Kb
0.3 Mb
3.6 Mb
3.7Mb
p.Phe508
CFTR
CFTR
For PGD for single gene disorders, there are probs with using a direct mutation test in PGD - low quantity of DNA, risk of contamination, False +ves and -ves, allele drop out.
THEREFORE we use…
Whole genome amplification (WGA) and haplotyping. Looks at 15 highly polymorphic microsatellite markers within and around the gene, spanning 4 Mb either side of gene itself
Advantages of haplotyping - mutation indep, one test for all couples with that condn, multiple loci analysed simultaneously, tolerant of allele drop out
Misdiagnosis risk <2% but may be <0.1% per embryo replaced if all markers informative
D7S2502
D7S2460
IVS8CA
D7S2847
D7S480
1.7Mb
0.7Mb
11.3Kb
1.5 Mb
3.7Mb
MS19
0.4Mb

7. Results from D7S643 marker
Mother
Father
Affected child
Embryo

8. PGD assays that are set upCF
SMA
HD or 50% risk of HD
FraX
ADRP
As assay already set up, preparatory work-up for an individual family only takes 1-2 months from when all samples rec’d (which samples will be discussed later!)

9. PGD assays being set up next
DMD
Myotonic dystrophy
Beta thalassemia
VHL
For new conditions, allow up to one year for assay to be set up
Can take up to one year to set up new assay from when all the samples are rec’d. May be less than one year.

10. Criteria to receive funded PGD in Edinburgh
Known genetic risk
No unaffected child* with residency
Female age<40
Anti mullerian hormone (AMH) > or = 5
Adequate follicle count
Female BMI<30
Both partners must have “right of residency”
* If have unaffected child, can still have treatment but it would have to self-fund
Edin has rec’d NSD funding for 15 cycles of PGD for single gene disorders per year (April 2010-March 2011)
We have set up criteria to try to allow fair access to treatment and so we are not swamped with referrals.
1-2 cycles per couple are offered.
Ethics of who gets treatment and who does not? Who decides?
AMH measures ovarian reserve. Average AMH at 30yrs is 10 and at 40 yrs is 5.
* Cost of treatment is £4360 per cycle incl thawed embryo replacements arising from those cycles.

11. Levels of fertility with respect to AMH levels (pmol/L)
2.2
15.7
28.6
48.5
Very low
Low
Satisfactory
Optimal
High *
* associated with polycystic ovary syndrome or granulosa tumour
For our refs so far, AMH has ranged from 2.3 (2 couples so far have been declined tx as AMH<5) to (PCOS) . Average = 15.8 (or = 9.4 excluding the 2 cases of PCOS), Median = 8

12. Source of PGD referrals 24 couples referred Aug 09 - Nov 10
The geog spread of referrals as we would expect from the density of popln in each area

13. Referred by…
As expected, vast majority of pts ref’ed by local clin gen dept
Only one couple was not known to Clin Gen when they were referred (beta thal, subfertility)

14. For which condition?
Whilst CF and HD make up the majority of refs so far, we have been approached about a wide range of conditions
It’s labour-intensive setting up new assay for each condition - giving up to one yr timescale and
- also making most of two-way sharing of assays/info between Guy’s and Edinb

15. Age of female partner
Success of IVF treatment is strongly influenced by maternal age, the younger the better!
We currently don’t treat anyone aged 40 and over as PGD is unlikely to achieve an ongoing pregnancy for these women

16. Pilot study update Three CF couples have completed a treatment cycle.
One couple having second cycle
One couple having second thaw cycle
One couple delivered 14 Nov 2010!

17. Looking forward 3 couples starting treatment Dec 2010 (FraX, HD, CF)
4 couples ready to start early in New Year (50% risk HD, HD, CF, ADRP)
50% risk of HD individuals offered PGD by embryo exclusion (like fetal exclusion) PGD i.e. embryos at 50% risk are not replaced.
cf Non-disclosure PGD (where indiv is tested and result held by lab, IVF carried out and embryos tested or not tested as appropriate. Couple just told that the embryo being replaced is not at risk of HD). This is NOT currently offered in UK.
ADRP - new assay had to be developed using phase analysis from single sperm and new licence obtained from HFEA for this condn which takes up to 4 months to hear back

18. New referral form (Nov 10)

19. What to expect?
V involved process. Lower chance of ‘take home’ baby than trying themselves. Fertile couples don’t respond any better to drug stimulation
1-2 months bet referral and 1st appt (monthly clinic)
1-2 months til 1st IVF appt (which is a monthly clinic and it dep on when work up completed)
1 month til nurse appt to teach drug administration and discuss scheduling
Completing many consent forms req’d
Scheduling - spread out from other cycles and timing also to suit couple
Only single embryo transferred

20. What samples are needed to offer PGD haplotyping?
Need a ‘triplet of samples’
Dominant condition = affected individual and their parents. Partner also helpful.
X-linked condition = couple and affected child or affected parent
Recessive condition = couple and affected child (or both set of parents of couple if no affected child in family)
Need new blood sample from affected child. Mouthwash or working on Guthrie spot not feasible
With ADRP performed phase analysis on sperm as parental samples not avail.

21. Thank you Enquiries welcome Sally.Morton@luht.scot.nhs.uk
Tel:
6-weekly PGD team meeting
Any questions?
Thank you to all my colleagues and to you for listening!
Any questions?