Single Gene Testing. Using Preimplantation Genetic Diagnosis

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1 Single Gene Testing Using Preimplantation Genetic Diagnosis

2 Single gene testing using preimplantation genetic diagnosis (PGD) is a highly sophisticated scientific technique, whereby embryos can be tested for the presence or absence of a specific gene. This allows embryos that are not affected by a specific disorder to be selected for embryo transfer & subsequent pregnancy. 2

3 Since its introduction, single-gene PGD has helped hundreds of couples conceive healthy babies. Many of these conceptions have been for couples with serious heritable diseases in their family. Our internationally recognised PGD program continues to lead the development of genetic testing in Australia. What is single-gene PGD? Many serious genetic diseases are caused by a mutation in a single gene. Preimplantation Genetic Diagnosis (PGD) is an option for couples where one or both of them are carriers of such a mutation. PGD can allow these couples to avoid passing the resulting disorder on to their children. A few cells are removed from embryos that have been created through IVF and these cells are tested for the presence of the mutation. In this way embryos that are unaffected by the disease can be selected for transfer back to the woman. This form of testing is quite different from the PGD techniques that are used to study chromosome number (see below). At present, it is not technically possible to do a comprehensive test for the chromosome number at the same time as single gene testing. Until now, testing of the embryos for single gene disorders has had to be done using a technique called PCR. This technique involves the preparation of individual short gene sequences for each patient to test for the presence of the mutation in the gene. This is a very accurate technique but has the significant drawback of requiring three to six months of preparation of the individual gene sequences. The Virtus Health PGD laboratory that is used by IVFAustralia, has recently introduced the new technique of Karyomapping that can replace the traditional PCR technique for the vast majority of patients requiring PGD for single gene disorders. 3

4 Preparation for single gene PGD Contact IVFAustralia Initial information on PGD process & cost estimate Jen Cox Public Liaison Coordinator or your local IVFAustralia Clinic Genetic consultation Detailed discussion about your case Clinical Geneticist Dr Debra Kennedy (left) Genetic Counsellor Dr Janan Karatas (right) Meet with your PGD Nurse Collection of samples for karyomapping assessment & preparation for IVF treatment PGD Nurse Karen Cain will undertake your orientation Karyomapping Assessment Study of DNA swabs (parents and other family members) affected by the disorder. Gene sequences are then compared to work up a genetic fingerprint for the mutation that causes the diseases. Cells can then be removed from embryos to work out which will be affected or not by the disorder, or will be carriers that could pass it on. Fertility Specialist Consultation Plan your IVF cycle Fertility Specialist A/Prof Gavin Sacks (left) Fertility Specialist Dr Jeffrey Persson (right) Administration Appointment Detailed explanation of costs of PGD testing & IVF treatment Patient Support Team IVF cycle with single gene PGD 4

5 Who should consider single gene PGD? Patients who are at increased risk (eg 1:4) of having a child with an inherited genetic condition (for example, Haemophilia, Thalassaemia, Cystic fibrosis). It is important to note that single gene PGD is only possible where the exact gene mutation, causing the problem, has been identified. There are many disorders where, although it is apparent from the family history that the cause is genetic, the exact gene is simply not known. In this case, single gene PGD cannot help. As a consequence, a lot of the preparation for single-gene PGD involves detective work by the genetics team to obtain old records and samples of DNA from family members to track down the exact mutation and work out the genetic sequences involved. The essential choice that couples face in this situation are to either go through IVF having PGD with single-gene testing or to conceive naturally and have the pregnancy tested, then have to go through a termination of the pregnancy or to simply accept whatever outcome happens. This is a clearly a huge decision and it is discussed in more detail in this booklet. 5

6 What is involved in single gene PGD? To go through single gene PGD, a woman first has to go through an IVF cycle. This involves her taking injections of a naturally-occurring hormone called follicle stimulating hormone (FSH) to stimulate her ovaries to grow many more eggs than in a normal month. She also has to take medication to ensure that these eggs are not released before they can be collected for IVF. Sometimes this is a nasal spray and in other cases, it may be more injections. The eggs are then collected at a short procedure under general anaesthetic. a technique called intracytoplasmic sperm injection (ICSI). The resulting embryos (and not all eggs will fertilise) are grown in the laboratory for five days. Consultation with clinical Consultation with Feasibility testing may Consultation with your fertility geneticist & nurse to explain costs PGD nurse include blood, genetic & DNA testing specialist to plan your IVF cycle and sign forms Egg collection Sperm collection Intracytoplasmic sperm injection (ICSI) Fertilisation Day 3 embryo (8 cells) 6

7 The testing involves firstly, carefully removing one or two cells from each embryo laboratories in Sydney. The biopsied cells are then sent to our PGD laboratory for genetic testing. These are tested for the at-risk genetic conditions and genetically suitable embryos procedure that is usually similar to having a PAP smear performed. Studies to date have shown that embryo biopsy is safe and does not affect the ability of the tested embryo to continue normal growth and development. Day 5 Biopsy 1 or 2 cells Frozen embryo transfer Pregnancy blood test Fetal pregnancy scan at from embryo for PGD testing 2 weeks later 6 weeks Single gene PGD testing 7

8 What type of testing will be done? Your individual circumstances will determine which PGD test technique is most appropriate for your embryos, and the genetics team will discuss this with you. Common conditions being tested for Huntington disease (direct and exclusion) Cystic fibrosis Thalassaemia Duchenne muscular dystrophy Fragile X Hereditary breast/ovarian cancers (BRCA1/ BRCA2) Single gene disorders A gene is a sequence of DNA (genetic material) on a chromosome with a particular function (humans have up to 20,000 different genes, arranged on 23 pairs of chromosomes). When the DNA sequence on a particular gene is altered, a genetic disorder, such as Cystic fibrosis, may result. Karyomapping technology How does the new Karyomapping technology work? DNA samples are collected, usually via a simple blood or saliva test, from the couple as well as from other family members who are known to either carry or not carry the gene. Preliminary testing of these samples allows the DNA fingerprint in the region of the faulty gene to be determined. These DNA fingerprints will have a different pattern depending on whether they have come from a sequence of DNA with two normal genes, one faulty gene or two faulty genes. T sperm and a few cells are removed from each embryo for DNA fingerprint analysis. By comparing the DNA fingerprint of the embryos to the DNA fingerprint of the family members we are able to determine, with approximately 98% accuracy, which embryos have inherited the disease and which are free of the disorder. What else can Karyomapping detect? Our genetic material, or DNA, is tightly coiled into structures called chromosomes. Every cell in an embryo should have 46 chromosomes, arranged in 23 pairs. Sometimes embryos can have an extra or a missing chromosome. Embryos with an 8

9 extra or a missing chromosome normally either fail to implant or lead to an early miscarriage. Testing of the embryo for the number of chromosomes normally involves a quite separate genetic technique. However, sometimes the Karyomapping will be able to identify that an embryo has an abnormal number of chromosomes and, therefore, will not be viable. However, many chromosomal abnormalities are not apparent with Karyomapping. Just because the Karyomapping did not detect a chromosomal abnormality does not mean that the embryo necessarily has normal chromosomes. Will Karyomapping be suitable for me? All couples that are at risk of passing a single gene disorder to their children are potentially suitable for Karyomapping. We estimate that most couples with a single disorder will be able to have PGD with Karyomapping. We will do an early assessment to determine if Karyomapping might be suitable for you. This will involve an appointment with our clinical genetics team, including our clinical geneticist, as well as consultation with the Virtus Health laboratory team. Following this, we will need to collect a DNA sample from you and specific family members. Analysis of these DNA samples will take 2-4 weeks and will establish whether Karyomapping is appropriate. If this is the case, your IVF treatment can commence almost immediately. Patients for whom Karyomapping is not suitable will still be able to undergo PGD for their single gene disorder but will have to use the traditional PCR methodology. The PCR methodology requires that individual gene sequences are developed that can be matched with the exact mutation that the parents are carrying. The preparation of these individual gene sequences requires a further three-six months of preparation as well as the additional costs of preparing the gene sequences. However, if you have already paid for the karyomapping assessment, this cost will be discounted from the total cost of preparation of the PCR gene sequences What happens during the actual IVF cycle? During the IVF cycle, you will receive injections of female hormone (FSH) to stimulate your eggs to grow. You will then go through a short procedure under general anaesthetic to collect the eggs. The eggs will taken to the IVF laboratory and will be fertilised with your pa laboratory for five days. On the third day of growth, a laser will be used to create a small hole in the embryo After five days, a few cells will be obtained from each embryo and will be sent to the PGD laboratory for testing. Do be aware that the number of embryos that will be available for testing can be very variable. At IVF, an average of 10 eggs may be collected of which about six (on average) fertilise. Not all embryos grow well and an average of 2-3 embryos are 9

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11 commonly available for biopsy. However these numbers are only averages. Some women will have more eggs and embryos than this while other women will have fewer. Once the embryos have been biopsied, all the embryos, that are suitable for freezing (possibly including some that have not been biopsied), will be cryo-preserved for later transfer. The results from the genetic testing will be available within 10 days of the biopsy being performed. Your doctor and the genetics team will make arrangements to meet with you to go through the results. Once the genetic testing results are available, you can discuss the next step with your doctor. If there is a genetically normal embryo available, your doctor can make arrangements to have this transferred. A frozen embryo transfer cycle normally involves a fee for the cycle. At IVFAustralia, where all the embryos have been frozen, we will provide the first frozen embryo transfer cycle with no out-of-pocket costs for the cycle. Does the testing involved in preimplantation genetic diagnosis harm the embryo? Not as far as we know. Current research shows that the likelihood of a biopsied embryo implanting is exactly the same as a non-biopsied embryo. Despite the need for removal of a few cells from the embryo, there have been no reports of any health problems as a result of embryo biopsy in children conceived after PGD. Gender selection Some genetic conditions affect one gender, for example haemophilia and muscular dystrophy. Sometimes it is not possible to detect the exact genetic error that causes the disease. However, a different genetic technique (Array PGD) can be used to determine the gender of the embryos, and only embryos of the required gender and with the correct number of chromosomes will be transferred. It is very important to note that under Australian Ethical Requirements, we are only able to provide gender selection where there is a clear medical reason for doing so. In all other applications of PGD, we do not know the gender of the embryo. Exclusion testing Some people have (or have had) a parent affected by a serious genetic condition, such as Huntington disease, that does not generally manifest until later in life. Many people do not want to have testing to determine if they have inherited that condition from their parent, but at the same time do not want to risk passing it on to their own children. PGD can be used to perform exclusion testing where the genetic error itself condition. 11

12 What preparation is needed? Assessment by the IVFAustralia genetics team This is important to allow the team to investigate the exact gene that is involved in the problem. To do this, the team will need to gather all your previous test results, including any tests that have been carried out on other family members. They will take a full medical history from both partners as well as obtaining a very detailed family history. Additional DNA testing may be necessary to reach a precise diagnosis. It is very important to bring as much information as you have to these visits. The genetics team will spend time with you discussing your options and in particular the difficult question of whether to pursue single gene PGD or to consider alternative approaches as discussed below. The length of time taken for the assessment phase will depend upon how much genetic information is already available. Collection of samples will also be organised for karyomapping assessment. Review by a fertility specialist The fertility specialist will see you to discuss what is involved in IVF, will explain this process to you and will plan the details of your IVF cycle (which will include a sophisticated form of IVF known as ICSI). The IVF cycle itself The stimulation phase of the IVF cycle will last approximately two weeks or so. To ensure that your egg collection is synchronized with the day set for testing of your embryos, the genetics nurse and your fertility specialist will guide you in the preparation for your cycle. 12

13 Do I do single gene PGD or do I conceive naturally and test the pregnancy once it has implanted? This the huge decision that couples, who have been diagnosed as having a high risk of having a child affected by a genetic disorder, have to face. The table below sums up the pros and cons of the two approaches. Single gene PGD For Allows you to proceed into a pregnancy, confident that you do not have a child affected by the disorder Natural conception and testing of the pregnancy For The conception part is much easier and more straightforward The costs are significantly less Against The cost Single gene PGD can be very expensive. We will go through the costs with you in detail. The preparation time involved Identifying the specific genetic problem and developing the necessary genetic probes can take a long time. The uncertainty of IVF Although most couples carrying a single-gene disorder are fertile, IVF remains an uncertain process. Against The trauma of having to go through a termination of a much-wanted pregnancy This is all, clearly, a very personal and difficult decision indeed. Our genetics team will spend time with you going through every aspect of this and will guide you to the right choice for you. 13

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15 How much does PGD testing for single-gene disorders cost? An IVF cycle with PGD has three components of cost: 1 The cost of the IVF technique The normal costs of IVF. These include the normal fees associated with the cycle monitoring and IVF laboratory work. Current information about this component is available on the IVFAustralia website at 2 The cost of the hospital stays This includes the cost of the admissions, procedures and required anaesthetics for the egg collection and later embryo transfer. The exact amount of this varies depending on the hospital, the anaesthetist and whether or not you have private health insurance. 3 The cost of the PGD technology This is shown in the accompanying sheet. Our patient support team will be pleased to go through all of the costs that would be involved in your IVF cycle so that you are fully informed before you make any definite decisions to proceed. 15

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