Pre-implantation Genetic Diagnosis (PGD)

By Solmaz Sabeghi Medical Genetics Laboratory of Dr.Zeinali

Titles:

1. PGD/PGS – FISH and PCR 2. PGD candidates 3. Cases we are able to diagnose with PGD 4. PGD procedure 5. Nested PCR 6. STR analysis 7. Haplotype mapping 8. Misdiagnosis in PGD

Who might consider PGD 1. Couples who are carrier of an inherited disorder and don’t want to experience medical abortion or have done medical abortion for several times.

2. An Infertile carrier couples who are candidate for IVF.

3. Couples who are carrier of an inherited disorder which has no permission for medical abortion in our country (deafness, breast cancer and …).

4. Couples who desire sex Selection. 5. Families who have an affected child and want to have non affected HLA matched child who can be a donor for their sibling when stem cell transplantation is the only treatment.

Cases we are able to diagnose with PGD in this laboratory

1. X linked diseases (DMD, Hemophilia and …) 2. Autosomal diseases (Thalassemia, PKU, Deafness, EB, CF and …) 3. HLA typing 4. Aneuploidies for chromosomes 13, 18, 21, X and Y

PGD (Pre-implantation genetic diagnosis)

Genetic analysis of a single cell from an eight cell embryo done in conjunction with in vitro fertilization (IVF) to determine if an embryo carries a specific gene mutation.

Two types of techniques are common:

1. chromosome “painting” (or FISH) using fluorescent probes specific for each chromosome.

In this method cells are fixated on glass microscope slides and hybridized with DNA probes which are specific for part of a chromosome, and are labeled with a fluorochrome. Currently, a large panel of probes are available for different segments of all chromosomes.

2. Molecular method: genetic testing for specific disease loci using Polymerase Chain Reaction (PCR).

Molecular method is usually based on using short tandem repeats (STRs) and direct sequencing. Analyzing STR markers located along the gene has been extensively used in PGD for single gene disorders to confirm direct mutation detection methods.

PGD Procedure: 1. Detecting or confirming the mutation.

(Only in case of diagnosing a disease.)

2. Analyzing all STR markers which are associated with the disease. 3. Tracing inheritance pattern in the family by comparing alleles in affected and unaffected individuals.

4. Choosing heterozygote STR markers. STR markers P M AC Comment

4.1SD7Col3D 381/381 389/389 389/381 Homozygote

D3Col7SU13.1 243/249 241/245 243/245 Informative

D3Col7SU21.7 317/321 321/329 321/321 One common allele

D3Col7SU25.6 284/292 284/292 292/292 two common allele

D3Col7SD16.4 388/392 396/396 392/396 Informative father

D3Col7SD17.1 305/305 301/305 305/305 Informative mother

19SD7Col3D 289/289 289/289 289/289 Homozygote

D3Col7SD19.4 345/349 343/357 349/357 Informative

5: Referring couple to IVF clinic to undergo a hormone therapy procedure.

6. IVF 6-1: Injection of human chorionic gonadotropin (hCG) and follicle stimulating hormone (FSH) to stimulate ovulation. 6-2: Monitor ovum maturation in the ovary using

.ultrasound and testing hormone level

6-3: Trans-vaginal aspiration using hollow needle to collect ovum on ovulation date (Puncture). 6-4: Obtain sperm from father and assess quality. 6-5: Combine eggs and sperm in vitro, using intra-cytoplasmic sperm injection (ICSI), if sperm is low quality.

6-6: Nurture embryo growth by incubating in medium containing various nutrients and hormones.

6-7: Removing a single cell after 3 days from an 8 cell embryo using a fine glass needle to puncture the zona pellucida and aspirate the cell (Biopsy). Each cell is called a blastomere.

6-8: Blastomeres which are containing a nucleus are gently collected in medium called cell lysis buffer and referred to laboratory. In PGD section in our lab

7. Samples are incubated in a thermal cycler for 35 minutes to dissolve blastomere’s nucleus and release DNA using special program.

8: Performing nested PCR on lysed samples for amplifying STR markers and specific exon which is associated with the disease.

9: Performing fragment analysis and direct sequencing method using ABI genetic analyzer.

10: Haplotype mapping to analyze polymorphic STR markers located along the gene and trace inheritance pattern in blastomeres.

U25.6 U21.7 U13.1 D16.4 D17.1 D19.4

286 317 249 388 305 345

294 321 245 392 305 349

294 321 245 392 305 349

294 321 245 392 305 349

294 321 245 392 305 349

286 329 241 392 301 349

286 329 241 392 301 349

294 321 245 392 305 349

286 317 249 388 305 345

286 329 241 392 301 349

M

M M

M

M 294 321 245 392 305 349

294 321 245 392 305 349

M M

11: Reporting results to the IVF clinic 48 hours after biopsy.

12: Transfer or freeze embryos on fifth day after fertilization based on couples decision.

13: Performing pregnancy tests after 3 weeks.

14. PND Carry out prenatal diagnosis on chorionic villus sample (CVS) or amniotic fluid (AF) to confirm the PGD performance.

Allele drop out

Most common phenomenon in single cell PCR is the allele drop out (ADO). It consists of the random non-amplification of one of the alleles present in a heterozygous sample.

Allele drop out phenomenon

Autosomal markers Marker A Marker B Marker C

P 248/256 256/248 256/256 M 252/260 260/256 260/256

Cell 260/ADO (248) 260/ADO (256)

256/256 256/ADO (248) 256/ADO (260)

256/256 256/256

256/ADO (260) 256/ADO (260)

X linked markers Marker A Marker A Marker B

P 248 248 248 M 252/260 252/260 260/248

Cell 260/Y 260/ADO (248)

248/ADO (260) 248/ADO (252)

248/Y 248/248

248/ADO (260)

Chromosomal crossover

crossing over is the exchange of genetic material between homologous chromosomes that results in recombinant chromosomes.

Crossover in cell 2

U25.6 U21.7 U13.1 D16.4 D17.1 D19.4 Comment

P 286/294 317/321 245/249 388/392 305/305 345/349 - M 286/294 321/329 241/245 392/392 301/305 349/349 - AC 294/294 321/321 245/245 392/392 305/305 349/349 -

Cell1 286/294 329/321 241/245 392/392 301/305 349/349 Carrier Cell2 286/286 317/329 241/249 392/392 301/305 349/349 Crossover

Cell3 294/294 321/321 245/245 392/392 305/305 349/349 Affected

PGD analysis requires testing for multiple closely linked markers

for mapping an accurate haplotype to detect recombination events and enable precise diagnosis.

U25.6 U21.7 U13.1 D16.4 D17.1 D19.4

286 317 249 388 305 345

294 321 245 392 305 349

294 321 245 392 305 349

286 329 241 392 301 349

M M

294 321 245 392 305 349

294 321 245 392 305 349

286 329 241 392 301 349

294 321 245 392 305 349

286 317 249 392 305 349

286 329 241 392 301 349

M M M 294 321 245 392 305 349

294 321 245 392 305 349

M M

Contamination in PGD As the amount of DNA is low in a single cell, many enhancers are used in PCR to improve efficiency of DNA amplification. Therefore in case of contamination, it will be amplified more than usual.

Thanks for your attention