Genes are chemical messages that instruct cells how to grow and perform the different chemical reactions necessary for life. There are more than 30,000 different genes and virtually every cell in the human body contains two copies of each. One copy of each gene is inherited from the Father and the other copy is inherited from the mother. It is very important for good health that every gene functions correctly; just one defective gene can result in serious disease. Because children acquire their genes from their parents it is possible for a defective gene to be passed from one generation to the next. This is why some diseases are said to ‘run in families’ affecting generation after generation.

Disorders caused by the inheritance of a single defective gene are known as monogenic diseases or single gene disorders. Monogenic diseases fall into two main categories. Firstly there are ‘recessive’ diseases, which do not produce any symptoms unless a defective copy of the gene is passed on by both the Mother and the Father. The second category is comprised of disorders that are said to be ‘dominant’, which only require one defective copy of the gene to be inherited in order to occur. Hundreds of different monogenic diseases, caused by errors in hundreds of different genes, have been discovered. Most of these disorders are very rare; however a few are relatively common. Well known monogenic diseases include cystic fibrosis, sickle cell anemia and Tay Sachs disease, which are recessive diseases, and myotonic dystrophy and Marfan syndrome, which are dominant.

GENETIC TESTING

In many cases the gene causing a monogenic disease can be tested to determine whether or not it is normal or defective. Tests of this type allow people to find out whether or not they carry a defective gene. Often such tests can be performed long before any symptoms of the disease have occurred. For example, genetic tests are often used during pregnancy to find out whether a fetus is affected by a specific genetic disease. If the fetus is found to be affected then the parents must make the difficult decision of whether to continue with the pregnancy or have a termination. An alternative method that aims to produce children unaffected by a genetic disease without any need for pregnancy termination is preimplantation genetic diagnosis (PGD).

PREIMPLANTATION GENETIC DIAGNOSIS (PGD)

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for people at risk of passing on an inherited disease to their children. The main benefit of PGD is that it maximizes the chance that a couple will have an unaffected pregnancy, greatly reducing the possibility that they will have to contemplate pregnancy termination. This is achieved by analyzing embryos before they have implanted in the womb, in other words before pregnancy has begun. PGD usually requires that the couple undergoes in vitro fertilization (IVF) treatment. This involves hormonal treatments that allow the collection of multiple eggs from the mother. The eggs are then fertilized using the father’s sperm and the resulting embryos are transferred to an incubator. After three days the embryos usually consist of a tiny ball of eight cells, known as blastomeres. One embryo cell (blastomere) is then removed (biopsied) from each embryo and subjected to genetic testing. If a blastomere is found to be unaffected by the inherited disease then the embryo that it was removed from will also be unaffected. Embryos that are revealed to be healthy can be transferred to the womb, ultimately producing unaffected babies.
At this time, IRMS offers PGD primarily for three specific single gene disorders – cystic fibrosis, Tay Sachs disease and myotonic dystrophy. It is important to note that some of our protocols are experimental and are supervised by the Internal Review Board of Reprogenetics.

Biopsy of Blastomeres
A blastomere is a cell from an embryo. To test the blastomere, an opening is made in the covering of the embryo during its third day of development when it consists of 8-10 cells. The blastomere is removed via aspiration with a pipette. The embryo is placed in an incubator while the cell is analyzed.

Analysis
The biopsied cells are analyzed using a technique called the polymerase chain reaction (PCR). Each cell contains a minute amount of DNA (the material from which the genes are made). PCR is used to amplify the DNA to a detectable level. Once amplification has been accomplished scientists can use a variety of techniques to screen an individual gene for abnormalities. Only embryos with cells that are unaffected by the inherited disease are transferred to the mother’s womb and consequently only unaffected babies should be born.

Using PGD techniques it is possible to detect changes in the genetic code that cause inherited diseases. This is possible even if the defects (mutations) affect just one letter of the 3,000,000,000 letter genetic code
	Image 1- An unaffected cell. Both copies of the gene have a normal DNA sequence.
	Image 2 – An affected cell. An additional green ‘peak’ can be seen superimposed on a normal DNA sequence. This indicates that one copy of the gene has an incorrect DNA sequence, while the other has a normal DNA sequence.

Testing of the biopsied cells destroys them because their membranes must be broken open to release the DNA. As such, one cannot use them for any other purpose or return them to the embryo.

Preliminary Analyses
For PGD of monogenic disorders, we require blood samples from both the prospective parents. In some cases we also collect cheek cells. This is accomplished very easily by rinsing some water around the mouth and then spitting it into a collection tube. The collection of blood and cheek cells allows preliminary analyses to be performed, which are essential to confirm that our PGD techniques will be applicable. The patient must provide us with a deposit that covers the cost of the preliminary work. Once the samples and deposit are received, preliminary testing can be performed within a few days.




ISSUES OF PREIMPLANTATION GENETIC DIAGNOSIS

The Risk Of Embryo Biopsy
While PGD is a relatively new procedure in IVF, the micromanipulation or biopsy techniques required to perform the procedure have been in use for many years. The risk of accidental damage to an embryo during removal of the cell(s) is very low, around 0.6%. Procedures such as intra-cytoplasmic sperm injection (ICSI), fragment removal and assisted hatching are all performed by making openings in the covering of the egg and none have been found to have other than positive effects on embryo development and implantation.

Removal Of Cells From The Embryo
No part of the future fetus will be lacking because of the removal of one or two cells from the embryo on the third day after fertilization. All the cells at this stage are said to be totipotent, literally meaning “all potential”. These cells have not differentiated yet and can form any part of the resulting fetus. The cells that are removed are simply replaced by the embryo. The procedure merely delays continued cell division for a few hours, after which the embryo reaches the same number of cells as before and continues its normal development. An unanswered question is whether biopsy affects the frequency with which embryos implant in the womb. The existing data is incomplete; however, any reduction in embryo implantation due to the effects of embryo biopsy seems to minor.

Misdiagnosis And Other Issues
The accuracy of PGD for monogenic disorders is approximately 95%. This means that the error rate is 5%. This figure includes normal embryos incorrectly diagnosed as affected and abnormal embryos wrongly diagnosed unaffected. Additionally, approximately 10% of embryos tested can not be diagnosed due to inconclusive results. In most cases inconclusive results are due to the failure of PCR to amplify the DNA to a sufficient level for the disease gene to be detected. Due to the small chance of misdiagnosis as well as the presence of unrelated problems such as chromosome abnormality (for example Downs Syndrome), which we do not test for, we recommend prenatal testing be carried out after a pregnancy is established.




CASES PERFORMED TO DATE

The preimplantation genetic diagnosis team has been involved in more than 2000 cases of PGD (mostly for chromosome abnormalities) up to August 2003.  These cases were performed either at the institute itself, or from samples sent to Reprogenetics for testing.




COST OF THE PROCEDURE

Please inquire at your Fertility Center as to the current fees for PGD. The PGD fees are in addition to the cost of in-vitro fertilization (IVF) and embryo transfer. The PGD fees include the cost of the chemicals and enzymes necessary for DNA amplification using PCR, analysis of amplified DNA and the biopsy procedure. Insurance companies do not cover the cost of PGD, but the fee will be waived if the patient enrolls in an ongoing randomized trial in which only 50% of patients will have PGD performed.

PERFORMANCE OF THE PGD PROCEDURE

Dr. Santiago Munné, PGD Program Director, Dr. Dagan Wells, Molecular Genetics Supervisor, Dr. Jacques Cohen, Scientific Director, have been involved in PGD since the start of the technique. Their work on PGD for aneuploidy and monogenic disease has been published in >150 scientific articles, a reduced list of which is provided below. Two papers obtained the Prize Paper of the Society for Assisted Reproductive Technology in the 50th (1994) and 51st (1995) Annual Meetings of The American Fertility Society. More recently their work received the General Program Prize at the American Society for Reproductive Medicine (ASRM) in 2000.




PGD FOLLOW-UP PROGRAM

All patients who achieve pregnancy after IVF with PGD are asked to participate in our follow-up program. Information regarding pregnancy, pregnancy outcome and child development will be gathered.



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MYOTONIC DYSTROPHY
Myotonic Dystrophy, also known as Steinert's disease or Dystrophia Myotonica (DM) is the most common form of muscular dystrophy, affecting roughly 1 in 8,000 people. The disease has a variety of symptoms including an inability of muscles to relax after contraction, respiratory problems, adverse reactions to anesthesia, cardiac disease, difficulty in swallowing, digestive problems, excessive sleeping and mental disorders. People with DM are more likely to develop diabetes and cataracts later in life. The extent to which these symptoms are manifest varies between individuals. Some individuals remain undiagnosed because their symptoms are so mild. However, at the opposite end of the spectrum infants with the most severe form of myotonic dystrophy often die shortly after birth. In many cases the disease displays an effect known as "anticipation", which means that the symptoms become progressively worse with each generation.

Myotonic dystrophy is a monogenic disease, caused by the inheritance of a single defective gene. Everybody inherits two copies of the myotonic dystrophy gene (one copy from each parent). The inheritance of one defective copy of the gene is sufficient to cause myotonic dystrophy, in other words it is inherited in a dominant fashion. This means that if you are at risk of transmitting a defective myotonic dystrophy gene on average 50% of your children will have the disease.

It is possible to test the myotonic dystrophy gene during pregnancy, thus revealing whether the fetus is affected with the disease. If the fetus is affected then the parents face the difficult decision of whether to continue with the pregnancy or have a termination. An alternative to prenatal diagnosis is to use preimplantation genetic diagnosis (PGD), a method that allows detection of myotonic dystrophy in embryos before they implant in the womb. The main purpose of this test is to allow patients to have children unaffected by a specific inherited disease, without having to contemplate termination of an affected pregnancy. We have developed state-of-the-art PGD tests for myotonic dystrophy that has been successfully applied resulting in the birth of unaffected babies.

To perform the PGD test it is first necessary for the parents to undergo in vitro fertilization (IVF). Using IVF a number of embryos are usually produced. The embryos are grown in an incubator for three days, by which time they consist of a small ball of about eight cells. At this point a single cell can be removed without harming the embryo. The cell can then be subjected to genetic analysis to determine whether it carries a defective copy of the myotonic dystrophy gene. If no defective myotonic dystrophy gene is detected then the embryo is diagnosed as unaffected. Unaffected embryos can be transferred to the mother’s womb and any resulting pregnancy will be unaffected.




CYSTIC FIBROSIS

Cystic fibrosis is the most common form of inherited defect affecting Caucasians (people of European descent) and currently affects approximately 30,000 people in the United States of America. Approximately one in 25 Caucasians carries a defective copy of the cystic fibrosis gene. Cystic fibrosis is a recessive disease, in other words the inheritance of two defective copies of the gene (one from each parent) is necessary to cause the disease. People with one defective cystic fibrosis gene and one normal cystic fibrosis gene are not affected by the disease, but are said to be ‘carriers’. If a man and a woman who are both carriers of cystic fibrosis have children then on average one child in four will inherit a defective gene from each parent and will therefore be affected by the disease.
Cystic fibrosis affects the mucus and sweat glands of the body resulting in the production of thick mucus in the breathing passages of the lungs. This leads to chronic lung infections. Additionally, mucus obstructs the pancreas, preventing enzymes from reaching the intestines to help break down and digest food. In males cystic fibrosis is also frequently associated with infertility.
It is possible to test the cystic fibrosis gene during pregnancy, thus revealing whether the fetus is affected with the disease. If the fetus is affected then the parents face the difficult decision of whether to continue with the pregnancy or have a termination. An alternative to prenatal diagnosis is to use preimplantation genetic diagnosis (PGD), a method that allows detection of affected embryos before they implant in the womb. The main purpose of this test is to allow patients to have children unaffected by a specific inherited disease, without having to contemplate termination of an affected pregnancy. Our team of experienced scientists has developed state-of-the-art preimplantation genetic diagnosis tests for cystic fibrosis disease.
To perform the PGD test it is first necessary for the parents to undergo in vitro fertilization (IVF). Using IVF a number of embryos are usually produced. The embryos are grown in an incubator for three days, by which time they consist of a small ball of about eight cells. At this point a single cell can be removed without harming the embryo. The cell can then be subjected to genetic analysis to determine whether it carries a defective copy of the cystic fibrosis gene. If no defective cystic fibrosis gene is detected then the embryo is diagnosed as unaffected. Unaffected embryos can be transferred to the mother’s womb and any resulting pregnancy will be unaffected.

Tay Sachs Disease
In its classical form Tay Sachs disease causes progressive destruction of the central nervous system and is fatal during childhood. The first symptoms of Tay Sachs usually begin to appear at about six months of age. Development slows, there may be a loss of peripheral vision, and the child exhibits an abnormal startle response. By about two years of age, most children experience seizures and diminishing mental function. As the disease progresses the affected child begins to lose physical and mental abilities and may experience difficulties swallowing and breathing. Ultimately, affected children become blind, mentally retarded, paralyzed, and unresponsive to their surroundings. In cases of classical Tay Sachs survival does not usually exceed five years.

Tay Sachs is a recessive disease, in other words the inheritance of two defective copies of the gene (one from each parent) is necessary to cause the disease. People with one defective Tay Sachs gene and one normal Tay Sachs gene are not affected by the disease, but are said to be ‘carriers’. If a man and a woman who are both carriers of Tay Sachs have children then on average one child in four will be affected with the disease, having inherited a defective gene from each parent. A person's chances of being a Tay Sachs carrier are significantly higher if he or she is of eastern European (Ashkenazi) Jewish descent. Approximately one in every 27 Jews in the United States is a carrier of the TSD gene.
It is possible to test the Tay Sachs gene during pregnancy, thus revealing whether the fetus is affected with the disease. If the fetus is affected then the parents must decide whether to continue with the pregnancy or have a termination. An alternative to prenatal diagnosis is the use of preimplantation genetic diagnosis (PGD), a method that allows detection of affected embryos before they implant in the womb. The main purpose of this test is to allow patients to have children unaffected by a specific inherited disease, without having to contemplate termination of an affected pregnancy. Our team of experienced scientists has developed a state-of-the-art preimplantation genetic diagnosis test for Tay Sachs disease.
To perform the PGD test it is first necessary for the parents to undergo in vitro fertilization (IVF). Using IVF a number of embryos are usually produced. The embryos are grown in an incubator for three days, by which time they consist of a small ball of about eight cells. At this point a single cell can be removed without harming the embryo. The cell can then be tested to determine whether it carries any defective copies of the Tay Sachs gene. If a normal copy of the Tay Sachs gene is detected then the embryo is diagnosed as unaffected. Unaffected embryos can be transferred to the mother’s womb and any resulting pregnancy will be unaffected.