Preimplantation Genetic Diagnosis (PGD) of a wide range of single gene disorders

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