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Preimplantation Genetic Diagnosis
SR Ghaffari MSc MD PhDM Rafati, MD, PhD
Reproductive Biotechnology Research Center Avicenna Research Institute
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
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
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
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, …)
The Most Common Indications (ESHRE)
ESHRE Report
Genetic Techniques
Genetic analysis:
PCR-based techniques
Hybridization based techniquesFISH Array CGH
Next Generation Sequencing
Amplification-based PGD
In the Past
Target specific optimization • Time consuming• Limited number of disorders• Limited genetic tests carried out on blastomeres• High failure rate
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
Our ExperienceAvicenna Infertility Clinic
(2012-2015)
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
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
Designing Several Primer Pairs
AAAGAGGGTAACTCATTAATAAAATAACAAATCATATCTATTCAAAGAATGGCACCAGTGTGAAAAAAAGCTTTTTAACCAATGACATTTGTGATATGATTATTCTAATTTAGTCTTTTTC
AGGTACAAGATATTATGAAATTACATTTTGTGTTTATGTTATTTGCAATGTTTTCTATGGAAATATTTCACAGGCAGGAGTCCAATTTTCACTCATCTTGTTACAAGCTTAAAAGGACTATGGACACTTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACTCTGTTCCACAAAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCCAAATGAGAATAGAAATGATTTTTGTCATCTTCTTCATTGCTGTTACCTTCATTTCCATTTTAACAACAGGTACTATGAACTCATTAACTTTAGCTAAGCATTTAAGTAAAAAATTTTCAATGAATAAAATGCTGCATTCTATAGGTTA
1F-239 2F-379
3F-160 4F-174
1F TCAAAGAATGGCACCAGTGT 1F TCAAAGAATGGCACCAGTGT3R CGCAGTGTTGACAGGTACAA 4R TTCTCATTTGGAACCAGCGC
CFTR-Ex20-13
304bp
CFTR-Ex20-14
321bp
2F AATGACATTTGTGATATGAT 2F AATGACATTTGTGATATGAT3R CGCAGTGTTGACAGGTACAA 4R TTCTCATTTGGAACCAGCGC
CFTR-Ex20-23
265bp
CFTR-Ex20-24
282bp
3F ATTTCACAGGCAGGAGTCCA 3F ATTTCACAGGCAGGAGTCCA4R TTCTCATTTGGAACCAGCGC 2R CTTAAATGCTTAGCTAAAGT
CFTR-Ex20-34
177bp
CFTR-Ex20-32
276bp
Whole Genome Amplification
Rule out of Contamination
STR Markers
Fetus and the Parents
• M
Examples
Cystic Fibrosis
AAAGAGGGTAACTCATTAATAAAATAACAAATCATATCTATTCAAAGAATGGCACCAGTGTGAAAAAAAGCTTTTTAACCAATGACATTTGTGATATGATTATTCTAATTTAGTCTTTTTC
AGGTACAAGATATTATGAAATTACATTTTGTGTTTATGTTATTTGCAATGTTTTCTATGGAAATATTTCACAGGCAGGAGTCCAATTTTCACTCATCTTGTTACAAGCTTAAAAGGACTATGGACACTTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACTCTGTTCCACAAAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCCAAATGAGAATAGAAATGATTTTTGTCATCTTCTTCATTGCTGTTACCTTCATTTCCATTTTAACAACAGGTACTATGAACTCATTAACTTTAGCTAAGCATTTAAGTAAAAAATTTTCAATGAATAAAATGCTGCATTCTATAGGTTA
1F-239 2F-379
3F-160 4F-174
1F TCAAAGAATGGCACCAGTGT 1F TCAAAGAATGGCACCAGTGT3R CGCAGTGTTGACAGGTACAA 4R TTCTCATTTGGAACCAGCGC
CFTR-Ex20-13
304bp
CFTR-Ex20-14
321bp
2F AATGACATTTGTGATATGAT 2F AATGACATTTGTGATATGAT3R CGCAGTGTTGACAGGTACAA 4R TTCTCATTTGGAACCAGCGC
CFTR-Ex20-23
265bp
CFTR-Ex20-24
282bp
3F ATTTCACAGGCAGGAGTCCA 3F ATTTCACAGGCAGGAGTCCA4R TTCTCATTTGGAACCAGCGC 2R CTTAAATGCTTAGCTAAAGT
CFTR-Ex20-34
177bp
CFTR-Ex20-32
276bp
Single-cell Whole Genome AmplificationAssessment of DNA quality
Amplification of Specific Targets
Embryo, Heterozygous (carrier)
Embryo, Homozygous Mutation (affected)
Embryo, Wild Type (not affected)
Fibrodysplasia Ossificans Progressiva
Pedigree
Clinical Features
WGA
Amplification of Specific Targets
Embryo, Homozygous Mutation (affected)
Embryo, Wild Type (not affected)
Beta-thalassemia
Confirmation of the Detected Mutations
• The father
Confirmation of the Detected Mutations
• The father
Confirmation of the Detected Mutations
• The mother
Confirmation of the Detected Mutations
• The mother
STR Profiling
PGD بتاتاالسمیLinked 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
STR profiling
PGD بتاتاالسمیLinked 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
STR profiling
PGD بتاتاالسمیLinked 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
Embryo biopsy
Whole Genome Amplification
PCR
Investigation of EmbryosNormal
Embryo investigationNormal
Final report
• M
Rule out of contamination
Allele drop-out
Thanks for Your Attention
5/22/13Rafati M 64
