PGD, Single gene disorders

1. Preimplantation Genetic
Diagnosis
SR Ghaffari MSc MD PhD
M Rafati, MD, PhD
Reproductive Biotechnology Research Center
Avicenna Research Institute

2. History
 In the late 1980s several pioneers and their groups began
preimplantation genetic diagnosis (PGD) with the detection
of single gene disorders
 1990 :The first successful PGD cycle was reported

3. PGD in Clinic
 By the mid-1990s there were still only 100 live born
children resulting from PGD
 For most of the next decade, relatively few clinical cases
were performed, and the field generally was considered
‘‘boutique’’ in nature
 By 2013 the mood has changed, PGD is no longer
boutique

4. What is PGD?
• The procedure which involves the removal of
one or more blastomeres to test for mutations
in a specific gene sequence or chromosomal
abnormalities before transferring

5. PGD Indications
• An alternative to conventional PND for couples with a
significant risk of transmitting a serious genetic disorder
to their offspring
• Avoiding abortion (enormous physical and psycological
burden)
• Presence of ethical concerns in aborting embryoes
affected with specific disorders ( for example: hearing
loss, skeletal disorders, …)

6. The Most Common Indications (ESHRE)

7. ESHRE
Report

8. Genetic Techniques
Genetic analysis:
PCR-based techniques
 Hybridization based techniques
FISH
 Array CGH
 Next Generation Sequencing

10. Amplification-based PGD

11. In the Past
Target specific optimization
• Time consuming
• Limited number of disorders
• Limited genetic tests carried out on blastomeres
• High failure rate

12. At Present
• Whole genome amplification of single cell
• Degenerate oligonucleotide primed PCR (DOP-PCR)
• GenomePlex Single Cell Whole Genome Amplification
• Multiple displacement amplification (MDA)
• A universal protocol for PGD of single gene disorders for a
wide range of genetic disorders
• Large amount of DNA from each blastomere (µg)
• No limitation in genetic investigations

13. Our Experience
Avicenna Infertility Clinic
(2012-2015)

14. Wide Range of Genetic Disorders
Investigated disorders till now:
 Beta-thalassemia
 Spinal Muscular Atrophy
 Duchenne Muscular Dystrophy
 Cystic Fibrosis
 Congenital Hearing Loss
 Achondroplasia
 Metachromatic Leukodystrophy
 Fibrodysplasia Ossificans Progressiva
Even rare
disorders

15. Workflow
 Genetic Counseling
 Mutation detection or confirmation
 Designing multiple primer sets
 STR profiling of the parents
 Whole genome amplification of biopsied single cells
 Subsequent investigations:
 PCR amplification
 Sequencing
 STR profiling

16. Designing Several Primer Pairs

17.  AAAGAGGGTAACTCATTAATAAAATAACAAATCATATCTATTCAAAGAATGGCACCAGTGTGAAAAAAAGCTTTTTAACCAATGACATTTGTGATATGATTATTCTAATTTAGTCTTTTTC
 AGGTACAAGATATTATGAAATTACATTTTGTGTTTATGTTATTTGCAATGTTTTCTATGGAAATATTTCACAGGCAGGAGTCCAATTTTCACTCATCTTGTTACAAGCTTAAAAGGACTATGGACAC
TTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACTCTGTTCCACAAAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCCAAATGAGAATAGAAATGATTTT
TGTCATCTTCTTCATTGCTGTTACCTTCATTTCCATTTTAACAACAGGTACTATGAACTCATTAACTTTAGCTAAGCATTTAAGTAAAAAATTTTCAATGAATAAAATGCTGCATTCTATAGGTTA
1F-239 2F-379
3F-160 4F-174
1F TCAAAGAATGGCACCAGTGT 1F TCAAAGAATGGCACCAGTGT
3R CGCAGTGTTGACAGGTACAA 4R TTCTCATTTGGAACCAGCGC
CFTR-Ex20-13
304bp
CFTR-Ex20-14
321bp
2F AATGACATTTGTGATATGAT 2F AATGACATTTGTGATATGAT
3R CGCAGTGTTGACAGGTACAA 4R TTCTCATTTGGAACCAGCGC
CFTR-Ex20-23
265bp
CFTR-Ex20-24
282bp
3F ATTTCACAGGCAGGAGTCCA 3F ATTTCACAGGCAGGAGTCCA
4R TTCTCATTTGGAACCAGCGC 2R CTTAAATGCTTAGCTAAAGT
CFTR-Ex20-34
177bp
CFTR-Ex20-32
276bp

18. Whole Genome Amplification

19. Rule out of Contamination

20. STR Markers

21. Fetus and the Parents
• M

22. Examples

23. Cystic Fibrosis

24.  AAAGAGGGTAACTCATTAATAAAATAACAAATCATATCTATTCAAAGAATGGCACCAGTGTGAAAAAAAGCTTTTTAACCAATGACATTTGTGATATGATTATTCTAATTTAGTCTTTTTC
 AGGTACAAGATATTATGAAATTACATTTTGTGTTTATGTTATTTGCAATGTTTTCTATGGAAATATTTCACAGGCAGGAGTCCAATTTTCACTCATCTTGTTACAAGCTTAAAAGGACTATGG
ACACTTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACTCTGTTCCACAAAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCCAAATGAGAATAGAA
ATGATTTTTGTCATCTTCTTCATTGCTGTTACCTTCATTTCCATTTTAACAACAGGTACTATGAACTCATTAACTTTAGCTAAGCATTTAAGTAAAAAATTTTCAATGAATAAAATGCTGCAT
TCTATAGGTTA
1F-239 2F-379
3F-160 4F-174
1F TCAAAGAATGGCACCAGTGT 1F TCAAAGAATGGCACCAGTGT
3R CGCAGTGTTGACAGGTACAA 4R TTCTCATTTGGAACCAGCGC
CFTR-Ex20-13
304bp
CFTR-Ex20-14
321bp
2F AATGACATTTGTGATATGAT 2F AATGACATTTGTGATATGAT
3R CGCAGTGTTGACAGGTACAA 4R TTCTCATTTGGAACCAGCGC
CFTR-Ex20-23
265bp
CFTR-Ex20-24
282bp
3F ATTTCACAGGCAGGAGTCCA 3F ATTTCACAGGCAGGAGTCCA
4R TTCTCATTTGGAACCAGCGC 2R CTTAAATGCTTAGCTAAAGT
CFTR-Ex20-34
177bp
CFTR-Ex20-32
276bp

25. Single-cell Whole Genome Amplification
Assessment of DNA quality

26. Amplification of Specific Targets

27. Embryo, Heterozygous (carrier)

28. Embryo, Homozygous Mutation
(affected)

29. Embryo, Wild Type (not affected)

30. Fibrodysplasia Ossificans
Progressiva

31. Pedigree

32. Clinical Features

33. WGA

34. Amplification of Specific Targets

35. Embryo, Homozygous Mutation
(affected)

36. Embryo, Wild Type (not affected)

37. Beta-thalassemia

38. Confirmation of the Detected Mutations
• The father

39. Confirmation of the Detected Mutations
• The father

40. Confirmation of the Detected Mutations
• The mother

41. Confirmation of the Detected Mutations
• The mother

42. STR Profiling
PGD ‫بتاتاالسمی‬
Linked STR Marker(s)
HBB-D11S4891 (75-100bp) Di nucleotide
Unlinked STR Markers
D18S535 (130-155bp) Tetra Nucleotide
FGFR3-D4S3038 (163-180bp) Di Nucleotide
93.2.3
7pair Primers
HBB-D11S1760
HBB-D11S1871
HBB-D11S4891
GJB2-D13S141
FGFER3-D4S3038
D18S535
D21S11

43. STR profiling
PGD ‫بتاتاالسمی‬
Linked STR Marker(s)
HBB-D11S1760 (75-100bp) Di nucleotide
Unlinked STR Markers
GJB2-D13S141 (115-…bp) Di Nucleotide
D21S11 (230-326bp) Tetra Nucleotide
93.2.3
7pair Primers
HBB-D11S1760
HBB-D11S1871
HBB-D11S4891
GJB2-D13S141
FGFER3-D4S3038
D18S535
D21S11

44. STR profiling
PGD ‫بتاتاالسمی‬
Linked STR Marker(s)
HBB-D11S1871 (160-200bp) Di nucleotide
93.2.3
7pair Primers
HBB-D11S1760
HBB-D11S1871
HBB-D11S4891
GJB2-D13S141
FGFER3-D4S3038
D18S535
D21S11

45. Embryo biopsy

46. Whole Genome
Amplification

47. PCR

48. Investigation of Embryos
Normal

49. Embryo investigation
Normal

50. Final report
• M

51. Rule out of contamination

52. Allele drop-out

53. Thanks for Your Attention
5/22/13Rafati M 64