PREIMPLANTATION GENETIC TESTING (PGT) FORDE NOVO MUTATIONS (DNM)

Abstract

Introduction PGT-M may presently be applicable for any inherited disorder for which sequence information or relevant haplotypes are available for the detection by direct mutation analysis or haplotyping in oocytes or embryos. However, these approaches cannot be applied in cases of de novo mutations (DNM) in parent(s) or affected children, as neither origin nor relevant haplotypes are available for tracing the inheritance of this DNM in single cells biopsied from embryos or in oocytes. On the other hand, with the improved awareness of PGT, an increasing number of couples request PGT, without any family history of the genetic disease that has been first diagnosed in one of the parents or in their affected children. So special PGT strategies are required for the genetic conditions determined by DNM. Materials and Methods We developed PGT strategies for DNM which were applied for 277 families with 83 different genetic conditions. The majority, 256 of them, were determined by dominant mutations, with only 4 by autosomal recessive and 21 - by X-linked DNM. It is of interest, that despite the expected predominance of dominant DNM of a paternal origin, with the increasing proportion of older paternal partners in the modern society, there was a comparable proportion of DNM of the paternal (138 couples) and maternal origin (110 couples). The latter presents a particular challenge in developing a PGT strategy, frequently requiring polar body testing to determine maternal haplotypes. The other challenge is presented by gonadal mosaicism detected in increasing number of PGT parents. In addition, up to 10% of the tested DNM (29 patients) were first detected in the affected children, with no evidence of detectable mutation in parents, despite the finding the corresponding mutant haplotype associated with normal allele. Results PGT strategies for these families were different depending on the origin of DNM, and included an extensive DNA analysis of the parents and affected children prior to PGT, with the mutation verification, polymorphic marker evaluation, whole- and single-sperm testing, and PB analysis in order to establish the normal and mutant haplotypes, without which PGT cannot be performed. In cases of DNM of paternal origin, the DNM was first confirmed on the paternal DNA from blood and total sperm, followed by single-sperm typing to determine the proportion of sperm with DNM and relevant normal and mutant haplotypes. For a higher reliability of testing, the relevant maternal linked markers were also detected, to be able to trace for possible shared maternal and paternal markers. In cases of DNM of maternal origin, DNM was first confirmed in maternal blood, and PGT was performed, when possible, by PB analysis, to identify the normal and mutant maternal haplotypes. Also, in order to trace the relevant paternal haplotypes, single-sperm typing was performed, whenever possible, for avoiding misdiagnosis caused by possible shared paternal and maternal markers. In cases of DNM-detected first in children, the mutation was verified in their whole blood DNA, followed by testing for the mutation in paternal DNA from blood, total and single sperm. So, in contrast to previous PGT practice, performing PGT for DNM required extensive preparatory DNA work before performing the actual PGT, with the additional tests including single-sperm analysis and the requirement of performing sequential PB1 and PB2, followed by blastocyst analysis. Overall, we performed 516 PGT cycles for DNM for 277 couples, which resulted in pre-selection and transfer of 678 DNM-free embryos in 464 cycles (average of 1.4 embryos per transfer) yielding 262 (56%) unaffected pregnancies, with only 25 (9.5%) spontaneous abortions, and birth of 265 healthy children, confirmed to be free of DNM tested Conclusions This is the world's largest series of PGT for DNM, which could not be performed by traditional approaches, due to unavailability of family history and lack of any affected family member to identify the origin of mutation and trace the inheritance of the mutant and normal alleles in oocytes and embryos. However, as demonstrated the specific strategies may be developed in search for the possible origin of DNM and relevant haplotypes as the basis for developing a PGT design for each particular couple with DNM, allowing a highly accurate pre-selection of oocytes and embryos free from DNM.