Genetic Disorders and the Fetus. Группа авторов
particular challenge is also presented by PGT for mitochondrial diseases, which still cannot be done reliably. A novel approach has been made to transfer a nuclear genome from the pronuclear stage zygote of an affected woman to an enucleated donor zygote, or to transfer the metaphase II spindle from an unfertilized oocyte of an affected woman to an enucleated donor oocyte.72
As seen from Table 2.1, PGT is no longer restricted to conditions presented at birth; it is gradually expanding to include common diseases with genetic predisposition, such as cancers, performed in 10.5 percent of PGT‐M cycles, or nongenetic indications (7.2 percent of cases), such as PGT‐HLA with the purpose of stem cell therapy of affected siblings in the family.62
Here we discuss the application of PGT‐M to a wider range of disorders, including conditions determined by de novo mutations (DNMs), genetic predisposition for late‐onset disorders, and preimplantation HLA matching (Table 2.1).
De novo mutations
PGT is presently applicable to couples who, although they may themselves be noncarriers of a mutation, have been found to have a DNM in their gonads although there is no family history of the genetic disease, or the disease is first diagnosed in one of the parents or their affected children (see Figure 2.2). As neither the origin nor relevant haplotypes may be available for tracing the inheritance of such mutations in single cells biopsied from embryos or in oocytes, the main emphasis is on the identification of the mutation and/or relevant haplotypes enabling mutation detection. Accordingly, PGT strategies for DNM depend on their origin. DNA analysis of the parents and affected children prior to PGT is required for verification of the mutation and polymorphic markers through single sperm testing and polar body analysis, thereby providing the normal and mutant haplotypes to trace the mutation. If the origin of the mutation is paternal, confirmation is first sought on the paternal DNA from blood and total sperm, and then by single sperm typing to determine the proportion of sperm with DNM and relevant normal and mutant haplotypes. It is also useful to test the relevant linked markers for the partner, to exclude misdiagnosis due to possible shared maternal and paternal markers. Where the origin of the mutation is maternal, polar body testing is the method of choice, providing the normal and mutant maternal haplotypes. Again to exclude misdiagnosis caused by possible shared paternal and maternal markers, the relevant paternal haplotypes are established through a single sperm typing. If the mutation was first detected in children, both the maternal and paternal haplotypes are established as described.
The other important phenomenon detected in PGT for DNM is gonadal mosaicism, which can be detected in either parent. Although the strategies may differ depending on the type of DNM inheritance, the general approach involves the identification of DNM origin and search for a possible gonadal mosaicism and relevant parental haplotypes.
Despite the complexity of PGT for DNM, these strategies may be applied in clinical practice with extremely high accuracy without the traditional requirement for family data, which is not always available. Since the report of our first systematic experience of PGT for 152 families with different genetic disorders,73 we have performed 526 cycles from 283 couples for 81 different de novo conditions, resulting in 270 clinical pregnancies and the birth of 234 unaffected children, with no misdiagnosis.48
Late‐onset disorders
PGT for late‐onset disorders with genetic predisposition was first applied for a couple with inherited cancer predisposition, determined by p53 tumor suppressor gene mutations,74 which are known to determine a strong predisposition to many cancers. Traditionally, these conditions have not been considered as an indication for prenatal diagnosis that would lead to pregnancy termination, which is not justified on the basis of genetic predisposition. Rather, the possibility of choosing embryos free of genetic predisposition for transfer would obviate the need for considering pregnancy termination, as only potentially normal pregnancies are established. Although the application of PGT for these conditions is still controversial, it has been performed for an increasing number of disorders with genetic predisposition that present beyond early childhood and may not even occur in all cases, including inherited cancers and heart disease.6, 7, 25, 74–77
We have performed a total of 874 cycles for 56 different forms of cancers, the most frequent being breast cancer (284 cycles) caused by BRCA1 (159 cycles) and BRCA2 (125 cycles) mutations. A total of 199 PGT cycles for BRCA1/2 resulted in transfer of one or two embryos, yielding 131 pregnancies and birth of 134 children free from genes predisposing to breast cancer.48
The other largest group of cancers for which PGT was performed was neurofibromatosis type 1 and type 2 (NF1/2), for which 103 cycles resulted in transfer of 138 genetic predisposition‐free embryos in 88 cycles, 53 clinical pregnancies, and 55 children born free from genes predisposing to neurofibromatosis.
Other cancers representing frequent indications for PGT were different types of Fanconi anemia (83 cycles), colorectal cancer (52 cycles), tuberous sclerosis types 1 and 2 (44 cycles) (see Figure 2.2), familial adenomatous polyposis (42 cycles), multiple endocrine neoplasia (34 cycles), and retinoblastoma (31 cycles); under 30 PGT cycles were performed for various other cancers. Overall, 966 genetic predisposition‐free embryos were transferred in 634 cycles, resulting in 387 (61.0%) clinical pregnancies and birth of 407 children free from the risk of developing these cancers.25, 48
The other emerging PGT‐M indication has been inherited cardiac disease, for which 109 cycles were performed relating to 23 different diseases. The most frequent indications were familial hypertrophic cardiomyopathy, CMH4 (22 cycles), dilated cardiomyopathy, CMD1A (17 cycles), Holt–Oram syndrome, HOS (8 cycles), acyl‐CoA dehydrogenase very‐long‐chain deficiency, ACADVLD (6 cycles), familial hypertrophic cardiomyopathy 1, CMH1 (6 cycles), long QT syndrome 1, LQT1 (6 cycles) and Noonan syndrome 1, NS1 (6 cycles); PGT for another 16 cardiac conditions were performed in five or less number of cycles. Overall, 123 embryos free of genes predisposing to cardiac disease were transferred in 89 cycles (1.38 embryos per transfer on the average), resulting in 55 clinical pregnancies (61.7 percent) and birth of 54 children free from inherited predisposition to these cardiac diseases. If not prevented, many of these conditions may manifest despite presymptomatic diagnosis and follow‐up, with their first and only clinical occurrence being a premature or sudden death.78 The couples at risk for producing progeny with inherited cardiac disease usually request PGT prospectively, with no previous pregnancies attempted, given one of the partners being a carrier of the specific mutation. Many couples already going through IVF for fertility treatment may have questions about the implications of genetic susceptibility factors for offspring, and the appropriateness of using PGT in testing for susceptibility to inherited cardiac disease.25, 48
Of special interest are PGT indications for late‐onset disorders with inherited predisposition to neurological disorders, including neurodegenerative conditions. A total of 960 PGT cycles were performed for these conditions, including 610 for intellectual disability, 210 for Huntington disease, 42 for different movement disorders, such as torsion or myoclonic dystonia, nine for Alzheimer disease, and nine for Prion disease. As many as 1,110 unaffected or genetic predisposition‐free embryos were transferred in 738 cycles (1.5 embryos per transfer on average), yielding 412 clinical pregnancies and birth of 406 infants unaffected or free of the genes predisposing to the above conditions.48 Thus, PGT provides a nontraditional option for patients who may wish to avoid the transmission of a mutant gene predisposing to late‐onset disorders in their future children. Because such diseases that present beyond early childhood and even later may not be expressed in 100 percent of cases, the application of PGT for this group of disorders is still controversial. However, for diseases with