First systematic experience of preimplantation genetic diagnosis for de-novo mutations

Abstract

Standard preimplantation genetic diagnosis (PGD) cannot be applied for de-novo mutations (DNM), because neither origin nor relevant haplotypes are available for testing in single cells. PGD strategies were developed for 80 families with 38 genetic disorders, determined by 33 dominant, three recessive and two X-linked DNM. All three recessive mutations were of paternal origin, while of 93 dominant mutations, 40 were paternal, 46 maternal and seven detected in affected children. The development of specific PGD strategy for each couple involved DNA analysis of the parents and affected children prior to PGD, including a mutation verification, polymorphic marker evaluation, whole and single sperm testing to establish the normal and mutant haplotypes and PGD by polar body analysis and/or embryo biopsy. Overall, 151 PGD cycles were performed for 80 families, for which a specific PGD design has been established. The application of these protocols resulted in pre-selection and transfer of 219 (1.72 per cycle) DNM-free embryos in 127 (84.1%) PGD cycles, yielding 63 (49.6%) unaffected pregnancies and birth of 59 (46.5%) healthy children, confirmed to be free of DNM. The data show feasibility of PGD for DNM, which may routinely be performed with accuracy of over 99%, using the established PGD strategy.

Germline mosaicism is a pitfall in PGD for X-linked disorders. Single sperm typing detects very low frequency paternal gonadal mosaicism in a case of recurrent chondrodysplasia punctata misattributed to a maternal origin

“…Predictions of embryos available for transfer or not transferable are depicted on the right order to verify this maternal origin scenario, we could have performed polar body analysis, as it has been shown to be an efficient strategy. 10,11 However, in the present case, only eight metaphase II oocytes were obtained for this couple, making putative maternal very low-grade mosaicism difficult to detect. As only male embryos without the causative variant were obtained following PGD, we decided to carry out an extensive single sperm analysis, which definitely demonstrated a paternally derived gonadal mosaicism.…”

“…For PGD purpose, we performed segregation analysis with linked markers and assigned the different haplotypes in this family based upon the clinical report associated with the referral. In order to verify this maternal origin scenario, we could have performed polar body analysis, as it has been shown to be an efficient strategy . However, in the present case, only eight metaphase II oocytes were obtained for this couple, making putative maternal very low‐grade mosaicism difficult to detect.…”

Effects of laser polar-body biopsy on embryo quality

“…Originally designed for detection of maternally derived aneuploidies and translocations (3), with advancing work and research, new indications for PBB are being described seemingly daily (2,13,14). PBB has been considered to be safe with little or no effect on embryogenesis, and a small amount of published work has dealt with the effects that PBB has on the developing zygote (1).…”

Karyomapping and how is it improving preimplantation genetics?

“…Regarding de novo mutations, it may not be possible with karyomapping to establish which parental chromosome is linked to the defect therefore, mutation testing is essential in these cases. As with all PGD technology therefore, karyomapping does not a-priori detect new mutations [111].…”