Embryo Genetic Testing (PGD) Single Gene Testing Fact Sheet

What is a single gene disorder? Single gene disorders are genetic conditions which are caused by specific gene change/s in a person’s DNA. Single gene disorders are heritable and often run in families. Individuals with a family history of a single gene disorder may be at risk for passing the condition onto their children. Examples of single gene disorders include Cystic Fibrosis, Huntington Disease, Fragile X and Myotonic Dystrophy. What is PGD with single gene testing? PGD is a viable option for couples at risk of passing on a specific single gene disorder to their child. PGD can be used to screen IVF embryos for the single gene disorder prior to implantation, with the aim of distinguishing between unaffected embryos and affected embryos. Only unaffected embryos will be selected for transfer to the uterus. In addition to testing for the single gene disorder, it may be possible to analyse the chromosomes in the embryo (ie. chromosome screening). Some embryos can have an abnormal number of chromosomes (ie: missing or extra chromosome/s) due to errors in cell division in the developing egg, sperm or embryo. This is known as chromosomal aneuploidy. An aneuploid embryo will fail to implant, miscarry, or result in the birth of an affected child (eg: a child with Down syndrome). This additional testing will help ensure that the embryo that is selected for transfer has the best possible chance of developing into a healthy baby. Monash IVF has developed PGD tests for numerous single gene conditions, some of which are outlined in Table 1. The Monash IVF PGD team is highly skilled in developing new PGD technologies and is able to develop tests for other genetic disorders upon request. If the disorder you are interested in is not listed please contact Monash IVF for further information. Table 1: Tests have previously been developed for these single gene disorders Achondroplasia

Hypophosphatemic Rickets

Adrenoleukodystrophy

Infantile Batten Disease

Alagille Syndrome

Incontinentia Pigmenti

Albright Hereditary Osteodystrophy

Juvenile Retinoschisis

Alpers Syndrome

KELL Blood Group

Alpha thalassaemia

Laing Distal Myopathy

Amyloidosis

Larsen Syndrome

Aniridia

Leri-Weill Syndrome

Antithrombin III

Lesch Nyhan Syndrome

Ataxia Telangiectasia

Lissencephaly

Autosomal dominant polycystic kidney disease

Loeys Dietz Syndrome

Autosomal recessive polycystic kidney disease

Long QT Syndrome

Becker Muscular Dystrophy

Lymphoproliferative Syndrome

Beta-Thalassaemia

Lynch Syndrome

Bethlem Myopathy

Marfan Syndrome

BrCa1

Medium Chain Acyl CoA Dehydrogenase Deficiency

BrCa2

Metachromatic Leukodystrophy

Bruton’s X-linked Agammaglobulinaemia

Multiple Endocrine Neoplasia, Type 1

Bullous Ichthyosiform Erythroderma

Multiple Endocrine Neoplasia, Type 2a

Cadasil

Multiple Osteochondromas (Exotoses)

Cardiomyopathy

Myotonic Dystrophy

Carpenter syndrome

Myotonic Dystrophy Type 2

Cartilage hair hypoplasia dysplasia

Myotubular Myopathy

Central core disease

Neurofibromatosis, Type 1

Charcot-Marie-Tooth, Type 1A

Neurofibromatosis, Type 2

Charcot-Marie-Tooth, Type 1B

Neurofibromatosis, Type 3

Charge syndrome

Ocular Albinism Type 1

Chronic Granulomatous Disease

Optic Atrophy

Congenital Amegakaryocytic Thrombocytopenia

Opitz GBBB Type 1

Congenital insensitivity to pain

Osteogenesis Imperfecta

Crouzon Syndrome

Peutz-Jeghers Syndrome

Cystic Fibrosis

Phaeochromoctoma

Deafness (connexin 26)

Polycystic Kidney Disease

Diastrophic Dysplasia

Pontocerebellar hypoplasia

Duchenne Muscular Dystrophy

Presenilin 1

Elastin gene disorder

Propionic Acidemia

Escobar Syndrome

Retinitis Pigmentosa

Facioscapulohumeral muscular dystrophy

Retinoblastoma

Familial Adenomatous Polyposis

Rhesus D Blood Group

Familial Hemophagocytic Lymphohistiocytosis

Saethre Chotzen Syndrome

Familial Motor Neuron Disease

Severe Combined Immune Deficiency

Fragile X

Smith Lemli Opitz Syndrome

Gastric Cancer

Spinal Muscular Atrophy (SMA)

Glycogen Storage Disease Type IV

Spinocerebellar Ataxia, Type 1

Haemophilia A

Spinocerebellar Ataxia, Type 2

Haemophilia B

Spinocerebellar Ataxia, Type 3

Hereditary Diffuse Gastric Cancer

Spinocerebellar Ataxia, Type 6

Hereditary Multiple Exostoses

Spinocerebellar Ataxia, Type 7

Hereditary Non-Polyposis Colorectal Cancer

Tay Sachs Disease

Hereditary Sensory Neuropathy

Treacher Collins Syndrome Type 1

Holt-Oram Syndrome

Trimethylaminuria

Huntington’s Disease

Tuberous Sclerosis

Hydrocephalus

von Hippel-Lindau Disease

Hypertrophic Obstructive Cardiomyopathy

Wiskott-Aldrich Syndrome

Hypohidrotic Ectodermal Dysplasia

Zellweger Syndrome

Hypophosphatasia

How is PGD with single gene testing done? Step 1: Genetic testing One or both partners must have had previous genetic testing to determine the exact gene change/s causing the genetic condition in their family. If the causative gene change/s has not yet been identified, the couple should be referred to meet with a clinical geneticist or genetic counsellor. These genetic specialists will be able to organise this genetic testing. The PGD team needs to know the causative gene change/s in order to proceed with developing a PGD test. Step 2: Genetic counselling in PGD clinic Once the specific gene change/s has been identified, the couple meets with a clinical geneticist or genetic counselor to discuss PGD. During this appointment the couple will be provided with information relating to the PGD process at Monash IVF and will have an opportunity to have any questions answered. If the couple wishes to proceed with PGD testing, the genetic counselor will arrange for the collection of blood/DNA samples for feasibility testing. Monash IVF currently offers single gene testing using two different test types, called SNP array testing and Polymerase Chain Reaction (PCR). The main difference between these two test types is that SNP array testing will allow simultaneous analysis of chromosome copy number, whereas PCR testing will only provide an analysis of the single gene disorder. The test type/s available to each couple will depend on the specific gene change/s that have previously been identified. Genetic counselling is an important step to help patients understand the differences between the tests, so that they make an informed decision regarding which test is best in their case. The PGD testing option that is performed will be decided by the patient in consultation with their IVF specialist and Genetic Counsellor. A comparison of the different PGD test types is provided in Table 1. Table 1: Detection capabilities of SNP arrays compared to PCR testing. Criterion

SNP array

PCR

Screen for targeted gene change/s







Screen 24 chromosomes




x

Confirm genetic parentage




Some

Detect extraneous DNA contamination




Some

Cost for feasibility testing







2-3 months

3-6 months

ICSI

ICSI

per cycle

per cycle

D3 or D5/6

D3 or D5/6

Frozen

Fresh (D3 biopsy) Frozen (D5/6 biopsy)

Estimated time frame for feasibility Fertilisation method (Standard insemination or Intracytoplasmic sperm injection)

Embryo biopsy fee charged Day of embryo biopsy Embryo transfer

Step 3: Feasibility testing Prior to commencement of an IVF/PGD cycle, it is necessary for the couple to undergo a feasibility test in order to determine if PGD will be possible for their particular gene change/s. The feasibility test will ensure that the final PGD test can adequately distinguish between unaffected embryos (suitable for transfer) and affected embryos (not suitable for transfer). Feasibility testing will require a copy of

the carrier’s genetic report/s and a blood sample from both partners. In most cases, a blood or DNA sample will also be required from one of the following: - Both partner’s parents - The couple’s child - A DNA sample from prenatal testing in a previous pregnancy In some cases, a sperm sample from the male partner may also be requested. These additional samples are used to track the inheritance of the particular gene change/s within the family. A feasibility report outlining the results of the feasibility testing is sent to the IVF doctor and genetic counsellor. The IVF doctor or genetic counsellor will contact the couple to go through the results of the feasibility test. In some instances it may not be possible to develop an accurate test and PGD may not be available. There is a non-refundable fee for the feasibility test. If feasibility has been confirmed, IVF/PGD may proceed. Step 4: IVF and embryo biopsy All couples undergoing PGD testing must undertake an IVF cycle to stimulate the woman’s ovaries to produce a number of eggs. These eggs are collected and fertilised using the male partner’s sperm. Embryos are created using a fertilisation method called Intracytoplasmic Sperm Injection (ICSI), which involves the injection of a single sperm into the egg. ICSI is specifically used in these cases to reduce the risk of misdiagnosis due to the presence of additional sperm around the egg/embryo. The resulting embryos are cultured in the laboratory and their growth is monitored on a daily basis. Embryo biopsy (sampling cell/s from the embryo for genetic testing) can be performed at two different stages during an embryo’s development: 1. Day 3 embryo biopsy is performed 3 days after fertilization, when the embryo is at the cleavage stage and is typically composed of 6 to 8 cells. Embryos that have developed to at least 5 cells on Day 3 are suitable for biopsy. A hole is drilled in the outer shell of the embryo and 1 or 2 cells are removed for genetic analysis (refer to Figure 1). Figure 1: Day 3 embryo biopsy

2. Day 5/6 embryo biopsy is performed 5 or 6 days after fertilization. By this time, the embryo should have developed to the blastocyst stage, and should be comprised of an inner cell mass (which will go on to form the fetus) and trophectoderm cells (which will go on to form the placenta). Embryos need to have a clear inner cell mass and a suitable number of healthy trophectoderm cells to be considered suitable for biopsy. Approximately 5 trophectoderm cells are removed for genetic analysis (Refer to Figure 2). Figure 2: Day 5/6 embryo biopsy

In the majority of cases, Monash IVF recommends Day 5/6 biopsy. This is due to the following reasons: 1. Randomised control trials have shown that Day 5/6 biopsy is better for the embryo compared with Day 3 biopsy (Scott et al, 2013).

2. The embryo has more cells on Day 5/6 of development (~100 to 150 cells) compared with Day 3 of development (~6 to 8 cells). This means: - More cells can be biopsied for genetic testing on Day 5/6 of development compared with Day 3 of development (ie: approximately 5 cells versus 1-2 1 2 cells, respectively). The availability of more cells improves the accuracy of the PGD test results. resul - Despite biopsying more cells from Day 5/6 embryos, a smaller percentage of cells is removed from the embryo following a Day 5/6 biopsy compared with a Day 3 biopsy (ie: we are removing approximately 5/100 (5%) cells on Day 5/6, compared with 1/6 (17%) cells on Day 3). 3. Day 5/6 embryos are more likely to be chromosomally normal then Day 3 embryos (as some of the embryos that are chromosomally abnormal on Day 3 do not have the developmental capacity to grow to Day 5/6 in culture). Therefore, growing the embryos to Day 5/6 before performing the PGD testing provides an element of natural selection. 4. Day 5/6 embryo biopsy enables the patient to confirm that their embryos are capable of developing to an advanced stage in culture before proceeding with PGD testing. The alternative is to perform PGD testing on Day 3 with the knowledge that the embryos may not continue to develop and therefore may not be suitable for transfer from an Embryology perspective. Step 5: Genetic testing Following embryo biopsy, the biopsied cells are transferred to a small test tube for genetic testing. The genetic testing process will depend on the PGD test type being performed: 1. SNP array (performed using “parental support”) In this procedure, the DNA from the embryonic cells is multiplied thousands of times (to generate enough DNA for testing) and is placed on a microarray platform. This microarray platform contains probes for over 300,000 different DNA sites. The DNA from the embryo biopsy sample e binds to the DNA probes on the microarray platform. Following binding, it is possible to “read” the DNA code at each of these DNA sites. By screening a blood sample from each partner in parallel with the embryonic cells, it is possible to determine whether each embryo is unaffected or affected by the specific single gene disorder. An analysis of all chromosomes is also performed to determine how many chromosome copies are present in each embryo (Figure 3). Only embryos which are identifed as being unaffected by the disorder, have the correct number of chromosomes and are developing normally will be considered suitable for transfer. Embryos which are affected and/or have an abnormal number of chromosomes (ie: missing or extra chromosomes) will not be considered suitable for transfer. Figure 3:: PGD testing method using SNP arrays.

+

Test cell/s

DNA from male partner

Test DNA

DNA from female partner

A

T

G

C

T

T

A

T

G

C

A T

A

T

G

C

A T

A

T

G

C

G C

A

T

G

C

G G

A

T

G

C

C C

A

T

G

C

A T

A

T

G

C

T A

A

T

G

C

A T

A

T

G

C

C A

A

T

G

C

A C

A

T

G

C

A A

A

T

G

C

G G

A

T

G

C

C G

A

T

G

C

C G

Chromosomes inherited by embryo

2. Polymerase chain reaction (PCR) In this procedure, the DNA from the embryonic cells is multiplied multipl ed thousands of times in a targeted manner using a procedure called PCR. This generates millions of copies of the gene of interest. The product from the PCR reaction is tested for the presence or absence of the

known parental gene change/s using a range of genetic techniques and a genetic analyser (Figure 4). In most cases, one or two linked markers are included in the final test in order to increase the accuracy of the results. Figure 4: Example of single gene PGD results. Affected gene copies are indicated by an asterisk.

c.2027 C to A gene change

D7S1825 linked marker

D7S1513 linked marker

Mother (normal)

Father (affected)

Embryo 1 (normal)

Embryo 2 (affected)

Step 6: Embryo transfer The time of embryo transfer will be dependent upon the PGD test type performed, as follows: 1. SNP array testing (performed using “parental support”) Because of the time taken to perform the genetic testing, the embryos must be frozen following biopsy. Final results are usually available 2 to 3 weeks after biopsy. If available, one or two unaffected embryos can be thawed for use in a frozen embryo transfer cycle. A PGD scientist/Embryologist will discuss the PGD results with the patient prior to transfer. The patient’s IVF nurse will organise a pregnancy test to be performed on Day 16 of the frozen embryo transfer cycle. This process should increase the chance of a healthy pregnancy and significantly reduce the risk of miscarriage. Surplus unaffected embryos will remain in storage. These embryos may be used in a subsequent cycle. Affected embryos will be removed from storage and allowed to succumb. 2. PCR testing Embryos identified as being unaffected by the disorder are considered suitable for transfer. • If a Day 3 biopsy has been performed, unaffected embryo/s can be transferred fresh on Day 5/6 of development. When a number of unaffected embryos are identified, morphological criteria are also used to determine the best embryo/s for transfer. Surplus unaffected embryos which are not transferred but which develop satisfactorily to the blastocyst stage may be frozen. These embryos may be used in a subsequent IVF cycle. Affected embryos will not be considered suitable for freezing and will be allowed to succumb. • If a Day 5/6 biopsy has been performed, the embryos must be frozen following biopsy. Final results are usually obtained within 1 week after biopsy. If available, one or two unaffected embryos can be thawed for use in a frozen embryo transfer cycle. Surplus unaffected embryos will be kept in storage. These embryos may be used in a subsequent IVF cycle. Affected embryos will be removed from storage and allowed to succumb. A PGD scientist/Embryologist will discuss the PGD results with the couple prior to transfer. The patient’s IVF nurse will organise a pregnancy test to be performed on Day 16 of the cycle. This process should increase the chance of a healthy pregnancy.

Why choose Monash IVF for PGD? Monash IVF has offered PGD as a clinical service since 1996 and is one of the few centres in Australia that specialises in this area of reproductive medicine. In 1996 we were proud to report the birth of Australia’s first PGD babies and since then we have performed over 3,000 PGD cycles with proven high success rates. Our specialised genetics team contains highly qualified experts in PGD, ensuring the best quality of care for patients. The genetics team at Monash IVF is responsible for providing a specialised PGD service not only to our own patients, but also to patients undergoing IVF cycles at fourteen different IVF clinics throughout Australia and New Zealand. While the main PGD laboratory is located in Clayton, Melbourne, Australia, embryo biopsy can be performed away from the genetics laboratory and the embryonic cells sent by courier to Clayton. Centralising the genetic testing enables patients to access the highest levels of expertise without having to leave their home state. Where can I get more information? If you would like further information regarding the PGD program at Monash IVF, please feel free to contact a member of the Genetics team on +61 3 9543 2833.