site. Following the nested amplification, PCR products are analyzed by restriction digestion, real‐time PCR, direct fragment size analysis, or mini‐sequencing. Depending on the mutation being studied, different primer systems are designed with special emphasis on eliminating false priming to possible pseudogenes, for which purpose the first‐round primers are designed to anneal to the regions of nonidentity with a pseudogene.25, 59
With the introduction of next‐generation technologies and the use of WGA prior to DNA analysis, the risk of ADO is further increasing, presenting even more problems in achieving accurate diagnosis.61 To improve the reliability of the test, the use of multiple linked markers became even more important, with importance of not only excluding the presence of the mutant gene, but also confirming the presence of the normal allele(s). Although a sufficient number of informative closely linked markers are usually available for multiplex PCR, this might not be the case in performing PGT by conventional PCR analysis in some ethnic groups.59 Currently available protocols allow an accurate PGT for complex cases, requiring testing for two, three, and even more different mutations.
PGT generally requires knowledge of sequence information for Mendelian diseases, but may also be performed when the exact mutation is unknown. With the expanded use of single nucleotide polymorphisms (SNPs), linkage analysis allows PGT for any monogenic disease, irrespective of the availability of specific sequence information.59–64 This is a more universal approach to track the inheritance of the mutation without actual testing for the gene itself, such as in karyomapping.65 On the other hand, a specific diagnosis is required for X‐linked disorders, which may be performed by polar body analysis to preselect the embryos deriving from mutation‐free oocytes which may be transferred irrespective of gender or the paternal genetic contribution.66
Polar body analysis (see Table 2.2 and Figure 2.2) also provides the prospect of pre‐embryonic diagnosis, which is required in many population groups where objection to the embryo biopsy procedures makes PGT nonapplicable. We performed the first pre‐embryonic genetic diagnosis for Sandhoff disease in a couple with a religious objection to embryo destruction.67 Although pre‐embryonic genetic diagnosis was previously attempted by first polar body testing,68–71 it is not actually sufficient for accurate genotype prediction without second polar body analysis, as shown in Figure 2.1. It is understood that for pre‐embryonic testing the second polar body analysis should be done prior to pronuclei fusion (syngamy), to ensure that only zygotes originating from mutation‐free oocytes are allowed to progress to embryo development and to be transferred, avoiding the formation and possible discard of any unaffected embryo.
A 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),
