PGT-M

PGT-M is a powerful tool in reproductive medicine, capable of screening for numerous hereditary conditions. These disorders result from mutations in individual genes. The particular disorders that PGT-M can identify are based on the specific genetic risks present in the parents.

Here are some examples of the types of disorders PGT-M can test for:

1. Cystic Fibrosis: A disorder affecting the lungs and digestive system, caused by mutations in the CFTR gene.
2. Sickle Cell Disease: A blood disorder caused by a mutation in the HBB gene, leading to abnormally shaped red blood cells.
3. Thalassemia: A blood disorder involving lower-than-normal amounts of hemoglobin, often due to mutations in the HBA1, HBA2, or HBB genes.
4. Huntington's Disease: A neurodegenerative disorder caused by mutations in the HTT gene.
5. Duchenne Muscular Dystrophy: A muscle-wasting disorder caused by mutations in the DMD gene.
6. BRCA1/BRCA2 Mutations: Associated with a high risk of breast and ovarian cancers.
7. Fragile X Syndrome: A genetic condition causing intellectual disability, more common in males, due to mutations in the FMR1 gene.
8. Tay-Sachs Disease: A fatal genetic disorder, more common in certain populations (e.g., Ashkenazi Jewish), caused by mutations in the HEXA gene.
9. Spinal Muscular Atrophy (SMA): A disorder leading to muscle weakness and atrophy, caused by mutations in the SMN1 gene.
10. Hemophilia: A blood clotting disorder, typically inherited in an X-linked recessive pattern, involving mutations in the F8 or F9 genes.
11. Marfan Syndrome: A connective tissue disorder, often due to mutations in the FBN1 gene.
12. Lynch Syndrome: A hereditary cancer syndrome associated with a higher risk of colon cancer and other cancers, caused by mutations in genes like MLH1, MSH2, MSH6, PMS2, and EPCAM.
13. Polycystic Kidney Disease: A condition characterized by the growth of numerous cysts in the kidneys, typically caused by mutations in the PKD1 or PKD2 genes.
14. Retinitis Pigmentosa: A group of genetic eye conditions that lead to progressive vision loss, caused by mutations in various genes.

Family studies have revealed that some types of cancers are hereditary. For some cancers, scientists have identified a specific gene mutation associated with the development of that cancer. It generally takes more than one gene mutation to cause cancer, but people with a single gene mutation may be at greater risk for developing the cancer.

For example, researchers believe that 1 in 10 cases of prostate cancer are inherited. However, In patients who are diagnosed before age 55, that rate rises to almost 5 in 10.

Recently, scientists identified a gene mutation that appears to be linked to inherited prostate cancer. However, the mutation is found in only a small percentage of patients with inherited prostate cancer. It is very likely that there are other gene mutations which are yet undiscovered. These undiscovered mutations may account for other cases of inherited prostate cancer.

Probably one of the best-known examples of a cancer that is linked to a gene mutation is breast cancer. Inherited mutations account for about 1 in 4 cases of breast cancer. A blood test is available that screens for two gene mutations called BRCA1 and BRCA2. These gene mutations account for 4% of all cases.

For families that are plagued by hereditary cancer, there were previously no methods available to help avoid passing cancer on to future generations.

By using PGT, prospective parents can elect to attempt pregnancy with embryos that do not have cancer causing mutations. Their children can then avoid the cancers that have stricken their families for generations. It is likely that these children would not be at any greater risk for these cancers than anyone in the general population.

Understanding Gene Mutations

Genes and DNA: Each chromosome contains thousands of genes. Genes are blueprints for creating proteins in cells, each of which has a different function in the cell. Genes are made up of a string of components called nucleotides. In human beings, there are four nucleotides, each designated by a letter. The four nucleotides are:

• Adenine A
• Guanine G
• Cytosine C
• Thymine T

The information in genes is encoded in the order of the nucleotides. For example, the sequence AGCTAGC would give different information than the sequence CATAGTA. When building a protein, the machinery inside the cell needs to know certain information such as:

• Where on the chromosome does the gene start?
• Which amino acids make up the protein and in what order do they go?
• Where does the gene end?

There are several abnormalities that can occur in genes:

Point mutation: This is when a single nucleotide has been switched. For example, in the spot where an “A” was supposed to go, instead, there is a “T." This type of abnormality can cause diseases like sickle cell anemia and cystic fibrosis. There two types of point mutations: missense mutations and nonsense mutations. A point mutation is a change in one nucleotide that results in the substitution of one amino acid for another in the protein made by a gene. A nonsense mutation is also a change in one DNA nucleotide, but instead of substituting one amino acid for another, the altered DNA sequence prematurely signals the cell to stop building a protein. This type of mutation results in a shortened protein that may function improperly or not at all.

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Deletion: This is when part or all of a gene has been deleted from the chromosome. The deleted DNA may alter the function of the resulting protein or eliminate it entirely. Common examples of diseases caused by gene deletions include Duchenne Muscular Dystrophy and retinitis pigmentosa.

Insertion: An insertion changes the number of DNA bases in a gene by adding a piece of DNA. As a result, the protein made by the gene may not function properly.

Duplication: A duplication consists of a piece of DNA that is abnormally copied one or more times. This type of mutation may alter the function of the resulting protein.

Frame shift mutation: This type of mutation occurs when the addition or loss of DNA bases changes a gene’s reading frame. A reading frame consists of groups of 3 bases that each code for one amino acid. A frame shift mutation shifts the grouping of these bases and changes the code for amino acids. The resulting protein is usually nonfunctional. Insertions, deletions, and duplications can all be frame shift mutations.

Repeat expansion: Nucleotide repeats are short DNA sequences that are repeated a number of times in a row. For example, a trinucleotide repeat is made up of 3-base-pair sequences, and a tetranucleotide repeat is made up of 4-base-pair sequences. A repeat expansion is a mutation that increases the number of times that the short DNA sequence is repeated. This type of mutation can cause the resulting protein to function improperly.

Genetic mutations may be consider to be “dominant” or “recessive” depending on the type of mutation and the disease. Mutations may be located on the sex chromosomes, X or Y, or they may be on the non-sex chromosomes, called autosomes.

PGT is indicated for couples at risk for transmitting a specific genetic disease or abnormality to their offspring. For carriers of autosomal dominant disorders, the risk that any given embryo may be affected is 50%, and for carriers of autosomal recessive disorders, the risk is 25%. For female carriers of mutations on the X chromosome, the risk of having an affected embryo is 25% (half of male embryos). PGT also can be elected by patients who carry mutations such as BRCA1 that do not cause a specific disease but are thought to confer significantly increased risk for a disease – in this case, breast cancer.

PGT for gene abnormalities

In order to analyze an embryo for a gene mutation via PGT, it is first necessary to understand what type of mutation is present and where it is located. In some cases, knowing the location of the gene mutation may be sufficient.

Since the amount of material we are studying is very small, it is first necessary to expand it so it can be tested more easily. This is done with a technology known as PCR (polymerase chain reaction). The DNA produced can then be analyzed by a variety of techniques such as restriction endonuclease digestion or “cutting” of the DNA into smaller segments followed by gel electrophoresis.

The major challenges of PGT relate primarily to the relatively short interval of time available for analysis and the fact that only one or two cells can be analyzed (compared with the hundreds of cells obtained via amniocentesis).

With PGT, misdiagnoses may result from:

• use of cell from the embryo which did not contain a nucleus
• failure of a segment a DNA to be identified
• external contamination

The risk for misdiagnosis relates directly to the type of genetic disorder for which testing is performed. The estimated risk of transferring an affected embryo mistakenly identified as normal by PGT is approximately 2% for recessive disorders and 11% for dominant disorders. The error rate can be significantly reduced if the PGT laboratory also analyzes areas of DNA that are close to the area where the mutation is located. This is called linkage analysis. Our laboratory performs linkage analysis routinely for PGT cases.

For couples known to be at risk for having children with inherited genetic disease, IVF with PGT represents a major scientific advance. Two professional fertility societies recommend counseling patients that patients that pregnancy rates may be lower than those achieved with IVF when PGT is not performed. Pregnancy rates after PGT may be reduced because genetic testing will decrease the number of embryos suitable for transfer. Embryos in IVF are typically chosen based on their appearacne under the microscope. However, if the best appearing embryos have genetic mutations then they cannot be used. The use of other embryos may lower the pregnancy rate.

Some of the cancers for which PGT has been performed:

Source: JAMA, December 13, 2006—Vol 296, No. 22

How is a gene mutation in an embryo found?

First, the couple wishing to conceive must complete blood testing. The parent whose family has hereditary cancer will be tested to see if they carry the cancer-causing gene mutation. Usually, blood from an already affected family member will be required. Both partners will have several DNA markers mapped and identified. These markers will be used to help identify what segments of DNA are present in the embryos.

If a gene mutation is present in the DNA of one of the members of the couple, a molecular probe is created that will be able to find and attach to the area of the chromosome where the gene mutation is located. If the gene itself has not been identified but the area where the gene is located is known, then markers on the chromosome close to the mutation can be used to identify affected embryos.

Using enzymes to cut DNA in certain, specific spots is performed. DNA pieces of different sizes can be produced and then separated. DNA which contains a gene mutation will result in pieces of different size to form and these can then be detected by analysis of the size of the DNA fragments.

PGT is performed during an IVF cycle. Pre-testing is performed on all couples that are going to undergo IVF. This includes assessment of ovarian reserve and uterine cavity, and semen analysis. Problems that may affect the likelihood for IVF to succeed are addressed before starting treatment.

Once the plan for identifying affected embryos is set, the couple will initiate an IVF cycle. The partner planning to carry the pregnancy will begin injectable fertility medications to stimulate the development and maturation of multiple eggs. They will be monitored during this time with blood tests and ultrasounds to follow the growth and development of the eggs in the ovaries.

When the egg reaches maturity, an egg retrieval will be performed, and at the same time, the semen specimen will be collected and prepared.

In the laboratory, the retrieved eggs are observed under a microscope. Each egg that has matured and appears healthy will be injected with a single sperm. this process is known as ICSI or intracytoplasmic sperm injection. The injected egg will then be placed in an incubator overnight. The next morning, the injected eggs are inspected to determine which ones have successfully fertilized, and then placed back into the incubator to continue growth and development.

Five to six days after the eggs are removed from the ovaries, embryo development is assessed. Embryos that have reached the blastocyst stage will undergo a trophoblast biopsy to remove several cells. The cells will be sent to the genetics laboratory for testing, and the embryo is frozen while waiting for the results.

If there are viable embryos that are not carriers for the cancer causing gene mutation, then these embryos may be used for embryo transfer. In some cases, it may be possible to perform other types of testing on the embryos, such as routine testing for abnormalities in the number of chromosomes. However, this may not be possible in all cases.

A frozen embryo transfer is performed by first preparing the uterus for implantation. This is commonly done by having the patient take estrogen and progesterone medications. Once the uterus is prepared, the selected embryo is thawed and the embryo transfer is performed. Eight days after the embryo transfer, the patient will return to our office for a pregnancy test. If pregnancy has been achieved, the early portion of the pregnancy will be monitored through visits to our office before we send the patient back to their regular obstetrician for routine obstetric care.