Practical Preimplantation Genetic Diagnosis

sampling (CVS) to be unaffected. Healthy unaffected twins were born, presently 7 years of age, with normal patterns of mental development.

The presented case is the first experience ofPGD for PKU in the experience of PGD for more than 100 different single-gene disorders, which were mainly performed for the at-risk couples with both unaffected partners who were heterozygous carriers of an autosomal recessive gene mutation, or one with a dominant mutation. Because, in the presented cases, it was a male partner who was compound heterozygous affected, the PGD strategy was based on the preselection of maternal mutation-free oocytes using a sequential PB1 and PB2 DNA analysis.

Based on the multiplex heminested PCR analysis, 6 of 11 oocytes with both PB1 and PB2 results were predicted to contain no mutant allele ofPHA gene. Four embryos resulting from these zygotes were transferred, yielding an unaffected twin pregnancy and birth of healthy children, following the confirmation of PGD by CVS. The high accuracy of this strategy was obvious from the follow-up study of the embryos resulting from the mutant oocytes, confirming the results of the sequential PB1 and PB2 analysis.

With progress in treatment of genetic disorders, PGD will have an increasing impact on the decision of the affected and well-treated patients to reproduce. For example, the life expectancy has been significantly improved for such common conditions as CFTR and thalassemias, which presently may be treated radically by stem cell transplantation. In each of such cases, the strategy depends on whether the affected partner is male or female, because testing may be entirely based on oocytes if the male partner is affected, in contrast to embryo testing when the female partner is affected. We have performed PGD for couples with affected partners with both of these conditions, presented below.

In thalassemia cases, there may be male factor infertility involved, so the male partners need appropriate treatments before PGD, or testicular biopsy has to be applied. This was the case in one of our couples with male thalassemic partner, when a sufficient number of oocytes with homozygous mutant or heterozygous PB1 was detected, but no appropriated sperm can be found for fertilization, so matured oocytes were frozen for possible future use after treatment. In the other two cycles, because the female partner was affected, blastomere biopsy was utilized, resulting in preselection of a single unaffected embryo for transfer in each cycle, yielding no clinical pregnancy. This was due to a limited number of oocytes obtained from the thalassemic female partners. However, in the well-treated patients, the situation maybe different as shown in the case with male affected partner with two different mutations, IVSI-110 and IVSII-745, and female partner with IVSI-110 presented in Figure 3.14. Of 25 embryos tested, 5 failed to amplify, 11 were mutant, and 9 heterozygous normal, providing a sufficient number of embryos for transfer.

We also performed PGD for two couples, one with female and one with affected male partner with CF. In a couple with maternal affected partner there were three different mutations in CFTR gene. The mother was affected with CF and had two different mutations, R117H and G542X (Fig. 3.15). The father was a carrier of 1717 mutation in CFTR gene. Paternal haplotypes were established using single sperm PCR, while maternal linkage was based on DNA amplification of PB1s. The couple had two previous pregnancies, the first one resulting in spontaneous abortion of twins, and the second pregnancy being terminated following prenatal diagnosis that identified an affected fetus with CF. So PGD was performed for three different mutations in CFTR gene, resulting in an unaffected pregnancy and birth of a healthy girl predicted and confirmed to be the carrier of maternal mutation G542X. It is of interest that multiplex PCR performed for three mutations in CFTR gene in this case was combined with age-related aneu-ploidy (see below) because of advanced reproductive age of the mother. Multiplex heminested PCR was performed on blastomeres from 14 embryos allowing simultaneous detection of the paternal and maternal CFTR haplotypes and nonsyntenic short tandem repeats (STRs) located on chromosomes 13, 16, 18, 21, 22, and XY. Six embryos were predicted to be carriers based on the presence of the maternal mutation and normal paternal CFTR gene. In addition to avoiding the transfer of the affected double heterozygous embryos, three aneuploid embryos were identified, involving aneuploidy for chromosomes 13, 18, 21, and X, and excluded from the transfer and freezing. Two unaffected embryos were transferred, resulting in pregnancy and birth of the healthy girl. DNA analysis of the newborn baby revealed the genetic profile identical to that of embryo #7, evidencing the usefulness and accuracy of the combined mutations analysis, linkage, and aneuploidy testing

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