Embryologist Media is pleased to count on the presence of Lidón Carretero, who works as a geneticist at Geniality (Madrid, Spain). This company has recently made it possible, for the first time in Spain, to assist in the birth of a baby girl without the mutation responsible for the breast cancer her mother had suffered from years before. Under normal circumstances, this girl would have had high genetic predisposition to manifest the disease, just like her mother. However, and thanks to scientific progress, the girl is now a reference for future similar cases that could also become free of that same cancer-causing mutation.

EM. Lidón, please tell us about the technique Geniality has performed in this particular case.

LC. Preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD) are complementary techniques to in vitro fertilization (IVF), whose aim is to screen every embryo for aneuploidies (PGS) and/or a specific monogenic disease (PGD). PGS is indicated in those cases in which the woman is older than 38, has a history of recurrent pregnancy loss, implantation failure or trisomy pregnancies, and also in couples carrying balanced chromosomal translocations. Issues on the male factor, such as sperm presenting altered fluorescent in situ hybridization (FISH) results, are another indication to perform PGS. This screening allows us to increase the chance of achieving a healthy pregnancy. On the other hand, PGD is indicated for fertile couples from families with a monogenic disease, aimed to avoid its transmission to the offspring.

EM. What are the steps to develop a PGD case?

LC. Genetic assessment consultation is the first step. This includes discussions about the genetic disorder in the families involved, the inheritance pattern and the characterization of the mutation. With all this information, the possibility of developing informativity testing (IT) prior to DGP is analyzed. There is also an IVF consultation in order to set the date when the woman should start ovarian stimulation. If the genetic disorder is included in the list of “accepted diseases”, IT development is granted. Otherwise, the whole study (IT+PGD) must be approved by the established Spanish Ethical Committee.

Once the IT confirms the mutation can be detected in embryos, ovarian stimulation may begin. Between ten and fifteen days after stimulation, biologists at IVF clinic collect the eggs. The number of eggs collected will vary, depending on the woman’s age and ovarian reserve. Eggs are fertilized in the IVF lab, and embryos can be biopsied either at cleavage or blastocyst stage. Biopsies are transferred to the genetics laboratory for PGD. Once the analysis is completed, we will know which embryos are transferrable (healthy) and which are not (carriers). Only one or two healthy embryos will be transferred into the woman, whereas the rest are cryopreserved.

Figure 1. Example of a molecular segregation study. In this case, the woman (represented by a circle) is affected by a genetic disorder. Her second chromosome carries the mutation responsible for the disease. Through PGD, only those embryos carrying the wildtype allele on the chromosome will be selected for transfer.

EM. Could you explain IT a little bit more in depth?

LC. The molecular strategy consists in co-amplifying multiple short tandem repeats (STR) markers linked to the mutant allele near the gene with the mutation, in order to map each mutant allele to each chromosome (Fig. 1). That is why it is necessary to have a genetic report with the characterized mutation since there are so many mutations that cause the same disease.

​Hands on the laboratory, a total of six of these STRs are selected, three upstream and three downstream the locus, so that the analysis is robust enough. All STRs selected must meet the following criteria: 1) maximum proximity to the target locus. In order to avoid recombination, it is advisable not to use markers further than 2Mb away from the locus; (2) enough heterozygosis, which means a high probability of having two different alleles. The more heterozygous the STR in the family is the more information it provides; (3) high repetition: tetraplets are normally easier to analyze than triplets, and triplets easier than duplets.

Once all STRs have been determined and located, primers for amplification must be designed. Primers to specifically amplify the mutant allele are also necessary. The SNaPshot technology allows finding out whether embryos carry the mutation. By doing so, it is possible to depict a segregation map for each allele, and only those STRs that are informative (different length for each allele in the carrier) will be used for PGD (Fig. 2).

“Since PGD entails certain ethical issues, each country has its own law and policies”.
Figure 2. Different combinations extracted from IT results for each STR. This information makes it possible to draw the segregation map.

EM. How long does the whole study take?

LC. IT results are available in less than one month. Ovarian stimulation lasts between ten and fifteen days. Egg fertilization and embryo development until biopsy takes another three or five days, depending on the embryo stage chosen to be biopsied. PGD is performed in a maximum of 48h, time for each embryo to be diagnosed. The woman will be transferred only one or two healthy embryos showing good morphodynamics parameters. Pregnancy can be confirmed about twelve days after transference. So that would mean roughly two months since the process begins until clinical pregnancy is confirmed. In case there is no healthy embryo or pregnancy has not been achieved, the couple is given the opportunity to try another cycle.

EM. In the case of Spain, not every single monogenic disease can be tested by PGD. What are the indications that make a specific genetic disease a proper target for PGD?

LC. Since PGD entails certain ethical issues, each country has its own law and policies. In Spain, these are regulated by the law 14/2006 (May 26th, 2006). PGD is indicated in the following scenarios: (a) the detection of serious, early-onset and incurable postnatal diseases, according to current scientific knowledge, in order to carry out the selection of non-assigned pre-embryos for transfer; (b) the detection of other alterations that may jeopardize the viability of the pre-embryo. Thus, each case must meet three key points: (1) monogenic disease with Mendelian inheritance and incomplete penetrance, (2) early onset, (3) severe morbidity and mortality. PGD for those monogenic diseases that do not meet all three requirements must be independently approved by the Spanish Ethical Committee for each couple.

EM. In this specific case, the application of PGD had to be approved by the committee. Which specific requirements were lacking, so that PGD could not be directly applied?

LC. 1.    Inheritance. Breast cancer due to mutations on the BRCA1 gene presents dominant autosomal inheritance, which means this woman’s children would have 50% probability of carrying the mutant. This condition shows incomplete penetrance, the probability of developing the disease being somewhere between 70% and 80%, which is considered as high.

2.    Onset. A disease is considered to be of early onset when it shows up before it is expected in the general population. For instance, cognitive impairment is usually first detected around the age of 60. This impairment is one of Huntington’s disease typical symptoms, and people affected by this disorder are only 40 years old, thereby Huntington’s disease is considered to be early onset. Most breast cancers and related deaths occur in women who are 50 or older. Nevertheless, the woman in our case was 29 when she was diagnosed with breast cancer, so the Ethical Committee took this fact into consideration in order to approve the PGD application.

3.  Morbidity and mortality. Breast cancer is the most common cause of cancer mortality among women. It usually manifests with an aggressive phenotype in young women, leading the death-causing cancers among women of ages 20-39.

As it has been explained, PGD in cases of breast cancer may not clearly meet some of the criteria required for direct application. Therefore, in this case, it was necessary to submit a formal request to the Ethical Committee.

EM. What kind of information may this request contemplate?

LC. This request should contain information about the disease (inheritance, clinical features and manifestation) and family history. It includes all medical reports, not only about the couple but also about affected relatives. Experts give much importance to family history. The more information on the family we provide, the stronger our request will be. Additionally, it must contain the bioinformatics strategy and the molecular protocol: characterization of the mutation and STRs to be used. The Ethical Committee looks over the request and makes a decision on whether it should be approved.

EM. In case the mutation is not characterized, what are the required steps to be considered prior to PGD?

LC. At first, we need to sequence the gene from the patient, to find out which specific mutation is present in the family. The probability of being inherited by her unaffected mother is very low, and since there were no other women affected in the family by the disease, we could not know whether the mutation had been de novo generated or inherited from her unaffected father. However, there are some genetic disorders, such as polycystic kidney disease, that can be caused by a mutation in any of the three different genes associated with the condition. In such cases it is quite possible that the patients ignore which gene has been mutated; the option here would be to perform a segregation study for those three genes to find out which one is responsible for the disease.

There are also cases in which one of the partners has relatives affected by a genetic disorder, but does not want to know whether he/she carries the mutation, since he/she may eventually suffer from the disease. In such cases, this person is not screened for the mutation. These are known as “exclusion cases”, and it is possible to perform an indirect PGD as long as a DNA sample from the affected relative can be obtained. By doing so, it can be inferred which chromosome of the relative contains the mutation, and embryos with that same chromosome can be discarded.

EM. It is known that cancer is not only associated with genetic causes, but it is also tightly linked to different modern lifestyle and environmental factors. This baby is now free from the mutation responsible for causing the type of breast cancer that her mother suffered from years ago but, could she still manifest this disease in the future?

LC. Through PGD we only study one or two mutations (depending on whether we have to deal with dominant or recessive inheritance), but there are indeed so many mutations that may cause the same genetic disorder. This woman’s children would have presented high probability (50%-70%) of manifesting this type of breast cancer due to her family genetic history, but thanks to PGD this probability is now as low as for any other individual from the population (12.5% in Spain). Unfortunately, we cannot eradicate genetic disorders from the population because we do not perform genetic engineering. We only select those embryos that are free of the mutation present in the family.


Embryologist Media wishes to give special thanks to Lidón Carretero and the entire team from Geniality, for sharing their knowledge and experience on the PGD approach, and for giving us the opportunity to spread this case to the community of ART specialists and the general public, as well.