due to ADO and preferential amplification in PB-based PGD.
The results of PB1 and PB2 analysis in another cycle performed for a couple with maternally derived mutation (13 bp deletion in APC gene), resulting in the birth of an unaffected child, was presented in Figure 3.24 above. Of 11 oocytes tested in this case, five were mutant, based on heterozygous PB1 and homozygous normal PB2 in three of them (oocytes #3, 5, and 9), homozygous normal PB1 and homozygous mutant PB2 in one (oocyte #4), and homozygous normal PB1 and PB2 in the other one (oocyte #7). The last pattern of the same homozygous genotype in both PB1 and PB2 maybe explained only by ADO of one of the alleles (ADO of the mutant allele in this case) in the corresponding PB1, because PB2 cannot contain the mutant allele without this allele being present in the oocyte following its maturation and the extrusion of PB1, which, therefore, should be heterozygous, corresponding to the oocyte genotype.
As seen from Figure 3.24, one of the oocytes tested had no information about PB2 and could not be genotyped (oocyte #11), while the remaining five oocytes were predicted normal (oocytes #1,2,8,10, and 12), ofwhich three resulted in embryos of acceptable quality and were transferred, yielding a singleton clinical pregnancy and birth of a healthy child following the confirmation by prenatal diagnosis.
The accuracy of PGD was, as usual, confirmed by the follow-up analysis of the embryos resulting from the mutant oocytes, as well as by prenatal diagnosis in the established clinical pregnancies and the blood test after delivery of a child. However, as shown in Figure 3.27, a cord blood sample was not appropriate for confirmation analysis, as it has been contaminated by maternal blood, clearly demonstrated by the presence of the mutant allele, as well as both maternal polymorphic markers in the cord blood sample. The fact that baby has no mutant allele was further confirmed by testing of a hill stick blood sample from the child, which contained no trace of the mutant gene, also in agreement with analysis of polymorphic markers. Therefore, the cord blood sample should not be material for confirmation of PGD.
One of the cycles performed for paternally derived Nt 482 A-G in exon 1 of VHL gene was presented in Figure 3.25 above. As seen from this figure, only one of four embryos in this cycle contained the mutant gene (embryo #9), the remaining three being normal. The haplotype analysis, an obligatory part of PGD for paternally derived dominant mutations, confirmed the presence of a normal paternal gene. As seen from the Figure 3.25, both embryos transferred in this cycle contained the polymorphic 123 bp marker, which is strongly linked to the normal paternal gene, therefore confirming the results of the mutation analysis. The transfer of these two normal embryos back to the patient, however, did not result in a clinical pregnancy. The affected embryo was reanalyzed, confirming the prediction of Nt 482 (A-T) mutation in the embryo.
The presented data demonstrate the acceptable diagnostic accuracy of both the PB and blas-tomere analysis for PGD of the above conditions. As shown by the follow-up analysis of the mutant embryos or those with insufficient marker information, the PGD results were confirmed in all resulting embryos available for the study, including 14 embryos in blastomere analysis and 48 embryos resulting from mutant ooocytes, detected by PB1 and PB2 analysis. The PGD diagnosis was also confirmed after birth of all 14 children, although as mentioned in the case of FAP, the use of cord blood for PGD confirmation should be avoided to avoid conflicting results due to contamination of cord blood by the maternal cells.
The data also show the need for avoiding mis-diagnosis due to ADO and preferential amplification in testing oocytes and blastomeres. The use of the multiplex nested PCR, involving simultaneous amplification of mutation and linked markers, as was discussed above, appeared to be efficient to avoid misdiagnosis , with every single marker result reducing the risk of misdiagnosis by half. For example, approximately half of the ADO in PB1, which was estimated to be in the range of 10%, maybe detected by applying the first linked marker, further picking up another 2-3% of ADO by the second marker, and reaching a complete detection rate with three markers. The same consideration maybe true for blastomere biopsy, however, taking into consideration an approximately double ADO rate in blastomeres compared with PB1 [24,27]. The other potential approach for the reduction of ADO rates may be the most recent application of a dual-probe real-time PCR, which according to our preliminary results may reduce the ADO rates by half, as discussed previously in Chapter 2.
As also seen from the presented results, indications for PGD are being further extended compared with the practice ofprenatal diagnosis, and
Figure3.27. Confirmation of PGD for APC mutation by cord and hill stick blood DNA analysis. (Left) Fluorescent PCR analysis of D17s559 polymorphic marker: Pick pattern (arrows) evidences the presence of both maternal alleles in cord blood, suggesting the maternal DNA contamination. (Right) Only normal allele was detected in hill stick (HS) and paternal (F) samples. Deletion in APC gene was detected in cord blood (CB) and maternal (M) samples. The presence of both maternal and a paternal alleles were confirmed by STR (HumFPS) and VNTR (PKU, D1s80) markers.
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