Cancer Predisposition

Using a standard IVF procedure, oocytes or embryos were tested for different mutations predisposing to cancer, preselecting and transferring only mutation-free embryos back to the patients. The procedure was performed for patients with predisposition to familial adenomatous polyposis coli (FAP), Von Hippel-Lindau syndrome (VHL), retinoblastoma, Li Fraumeni syndrome, determined by p53 tumour suppressor gene mutations, neurofibromatosis types I and II and familial posterior fossa brain tumour (hSNF5). Overall, 20 PGD cycles were performed for 10 couples, resulting in preselection and transfer of 40 mutation-free embryos, which resulted in five unaffected clinical pregnancies and four healthy children born by the present time. The data demonstrate the usefulness of this approach as the only acceptable option for at-risk couples to avoid the birth of children with an inherited predisposition to cancer, and to have a healthy child.

Technique:

PGD cycles were performed using a standard IVF protocol, coupled with micromanipulation procedures. For testing the maternal mutations, the first and second polar bodies (PB1 and PB2) were removed sequentially following maturation and fertilization of oocytes, while single blastomeres were removed from the 8-cell embryos for testing the paternal mutation.

In the couple at risk for VHL, the paternal mutation resulted from A to T substitution in nucleotide 482 of exon 1. Blastomere biopsy was performed in all three cycles for this couple, as well as for other couples with paternally derived mutations, including two couples with FAP, and couples with p53 tumour suppressor mutation and NF2. Maternal mutation in a couple at risk for familial posterior fossa brain tumour was due to G to A substitution in a donor splice site of exon 7, which alters the conserved GT sequence at the beginning of the intron, violating the GT rule for splice-site recognition. In this unique case, the mother was unaffected but her daughter, who inherited the mutation, had a brain tumour. Because the mutation was also detected in DNA from her uncle’s tumour, suggesting the risk of transmitting the mutation to the next child, PB1 and PB2 were removed in this case to preselect mutation-free oocytes in a standard IVF cycle.

This same approach was used in the couple with a maternal RB1 mutation; a bilateral RB found in the mother as a child, and also observed in her father. However, because the mutation in this particular case was unknown, PB1 and PB2 were removed and tested by linked markers for FAP, VHL, RD and hSNF5 brain tumor. DNA testing in all the PGD cycles was performed by multiplex nested PCR analysis, amplifying mutations simultaneously with linked markers both in the blastomere and PB1 and PB2, with sets of primers. Linkage analysis was performed for each couple, and the maternal and paternal haplotypes were established to avoid a possible misdiagnosis, which still requires a special attention because of the phenomenon of allele dropout (ADO) and preferential amplification, known to be frequent in a single cell DNA analysis. The patients gave consent, which was approved by Institutional Review Board, that, based on the multiplex mutation and marker analysis, unaffected embryos would be preselected for transferring back to the patients, while those predicted mutant would be exposed to confirmatory analysis using the genomic DNA from these embryos to evaluate the accuracy of the single cell-based PGD.

Treatment Value:

Overall, 175 oocytes or embryos were tested in 20 PGD cycles from 10 couples carrying the above-mentioned mutations (8.8 per cycle), resulting in preselection of 41 embryos free from mutations predisposing to cancer in all but one cycle (2.2 per cycle), which yielded five unaffected pregnancies (26.3% per transfer), and the birth of four mutation-free children (one stillbirth with confirmed mutation-free status, and one twin pregnancy ongoing). Twelve of these PGD cycles from six couples with maternal mutations were performed by sequential PB1 and PB2 analysis. Of 102 oocytes tested by both PB1 and PB2 DNA analysis (8.5 per cycle), 54 (4.5 per cycle) were predicted to be free from mutations predisposing to cancer. However, only 26 (2.2 per cycle) of these 54 mutation-free oocytes with sufficient linked marker information available resulted in cleaving embryos of acceptable quality for transfer, resulting in three clinical pregnancies and the birth of two unaffected children (one pregnancy ongoing).

These data demonstrate the acceptable diagnostic accuracy of both the PB and blastomere 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 the birth of 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. In conclusion, as seen from the presented results, indications for PGD are being further extended compared with the practice of prenatal diagnosis. Together with the previously reported application of PGD to p53 tumour suppressor mutations and NF1 and NF2, it is obvious that PGD for cancer predisposition is an acceptable approach for couples at risk, despite important ethical implications. Genetic counseling services may therefore consider informing patients at risk of having children with a strong genetic predisposition to cancers about the presently available option for PGD, without which these couples may remain childless because of their fear of having to choose prenatal diagnosis and possible pregnancy termination.

Next Steps:

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