PGD for single gene disorders 1. Presentation. What is it? Preimplantation genetic diagnosis (PGD) is a genetic analysis process linked to one IVF cycle. The analysis of embryos obtained by assisted reproduction techniques performed before the embryo implantation takes place includes: prolonged embryo cultivation, embryo biopsy, the genetic analysis of these biopsies and the transfer of normal embryos, as well as the vitrification (i.e. freezing) of the normal embryos not transferred. Single gene diseases are hereditary genetic pathologies that are caused by the alteration of DNA sequences in a specific gene. These mutations are generally transmitted in families, generation after generation but it is also possible that they could appear for the first time in the offspring of a couple or individual. 2. Objective. What is it used for? The PGD for single gene diseases aims to analyze a cohort of embryos in order to avoid the transmission of a hereditary disease present in the family to the offspring. This is achieved by classifying the analyzed embryos and separating those that have not inherited the mutation or mutations studied, hence fetuses developing from these embryos will not suffer from the disease(s) in question, whereas those with the mutation will be genetically abnormal. 3. Recommendations. Who and what is it for? This type of PGD is recommended for couples with personal or family backgrounds of a high risk of severe single gene diseases like cystic fibrosis, the fragile X syndrome, muscular atrophy/dystrophy, Huntington's disease, etc. 4. Advantages. What are its advantages over other tests? The main advantage of this type of genetic analysis is that it permits the study of various in vitro embryos in order to determine those that are genetically normal, therefore candidates for uterine transfer. This way couples with a high genetic risk can avoid having to make difficult decisions regarding interrupting abnormal pregnancies. 5. Samples. How? PGD for single gene diseases has two phases: (a) a genetic validation test and (b) genetic analysis of embryos.  The validation test is performed before the start of the assisted reproduction cycle and requires blood samples from the couple, and preferably, those of other family members relevant to the case. We distinguish between (i) an informative test, i.e. a more general genetic analysis using genomic DNA to check for the more common diseases such as cystic fibrosis, spinal muscular atrophy, Huntington's, etc. and (ii) a unique PGD genetic analysis protocol using a single cell in cases where less frequent Mendelian inherited diseases are likely to be present.  Regarding the embryo analysis, the biological sample obtained from the embryo biopsy is collected in a microtube in a sterile solution; it is then sent in a cold/frozen container to the analysis laboratory. Once received, the sample is maintained at ‐20°C until the required molecular genetics techniques are performed. 6. Shipping. How, when, where and who? The blood or mouth cell samples used for the genetic validation tests are sent at room temperature or refrigerated at 4°C.The embryo samples are transported frozen in a special in a well protected cold container (‐20°C) in order to avoid damage. They are sent immediately after biopsy. The delivery address is: IGENOMIX S.L. Laboratorio DGP monogénicas & diagnostico molecular www.igenomix.com Parc Científic Universitat de València C/ Catedrático Agustín Escardino nº9 46980 Paterna Valencia 7. Sample processing and results delivery. When? The informative validation tests are performed in a maximum of seven business days. The development of the protocol for single cell PGD requires approximately two months before the start of the assisted reproduction cycle. Regarding embryo analysis, the genetic testing and results delivery requires between 24‐48h.  Methodology.  Cellular lysis.  PCR amplification and fluorescent multiplexing  First round PCR multiplexing  Second round PCR  PCR product processing (restriction, mini sequencing, etc.)  Automatic analysis through capillary electrophoresis (CE). 8. How do I start? Get in contact with [email protected] or [email protected] 9. FAQs Is it necessary to have already identified the mutation or mutations that cause the pathology before performing a PGD for a single gene disease? It is very useful to have previously identified the mutation or mutations before PGD. Nevertheless where a precise clinical report is available and if the disease case is familial (various members of a same family are affected, preferably in different generations) it may be possible to perform an indirect analysis to offer PGD. Examples of this indirect strategy would be familiar cases of polycystic kidney disease; neurofibromatosis familiar type 1; Marfan familiar syndrome, etc. If it is a de novo case ‐ one with no previous family history of the disease ‐ it is essential to have identified the mutation that causes the disease prior to PGD. If a couple is fertile but has a background of genetic disease, can a PGD be performed? Even though PGD is linked to assisted reproduction, a fertile couple can equally use this genetic analysis in order to avoid having an affected offspring. This ensures gestation of a fetus free from the disease, and therefore avoids the difficult process of making decisions about interrupting abnormal pregnancies. If I have performed a PGD analysis in another center, do I have to perform the validation test again before the PGD cycle? We will probably have to repeat the validation test. This is because each center uses equivalent analysis strategies but these are not necessarily identical. For example, for each case multiple genetic markers are used to guarantee high diagnosis reliability: the markers chosen by one center and another are likely to be different therefore we will require different information from a new informative test. www.igenomix.com