Abstract Study question How to technically deal with the PGT-M set-up in case of de novo mutations in female or male affected patients with dominant disease due to de novo mutations. Summary answer PGT-M was performed for three couples carrying de novo mutations using direct and linkage analysis on sperm or polar bodies to define haplotypes and phase. What is known already Couples with a de novo mutation in a gene causing AD disease, such as FGFR3 (achondroplasia), NF1 (neurofibromatosis) and EXT2 (multiple exostosis) cannot undergo PGT-M via standard techniques like karyomapping, as the absence of affected relatives makes phasing impossible. However, linkage analysis combined with direct mutation analysis allows on haploid cells from the mutation carrier, such as sperm or polar bodies (PB), allows the correct association of a haplotype and the disease-causing mutation. Flanking informative STRs must be positioned at < 1 Mb of the gene, in order to minimize the risk of recombination during meiosis. Study design, size, duration Couples underwent pre-test counselling with a geneticist and an IVF specialist. Pathogenic variants were identified and their absence from the couples’ parents confirmed. Four to six informative STRs were identified. For males we analysed 20–50 isolated sperm to define the haplotypes and the phase, before starting with the stimulation cycle; for females, we needed to wait after the oocyte pick-up and the biopsy of PBs. Point mutations are identified by SNaPshot, deletions by multiplexed STS. Participants/materials, setting, methods The 3 couples in the study presented in IVF centres, requesting PGT-M for either male or female AD disease. They had genetic testing reports from other laboratories. For FGFR3 and NF1, the described variants were confirmed. The patient with multiple exostosis came with a negative genetic result for EXT1 and EXT2 genes, but after diagnostic-quality NGS (Blueprint Genetics, Finland) we identified an EXT2 deletion. Diagnostic multiplex PCR was then performed on embryos or polar bodies. Main results and the role of chance The setup started with the confirmation of the mutations in the 3 couples and the confirmation of the de novo status. Four to six informative STRs were then identified for each couple. Multiplex PCR containing the STRs and the SNAPSHOT analysis for the point mutations was developed. To identify the phase and the disease-carrying haplotype in male carriers, we performed a multiplex PCR on 20–50 spermatozoa. In the female patient with NF1, the haplotype and the phase were determined on the polar bodies; the mutation was on her paternal allele, as predicted genetically. Prior to PGT, we evaluated the robustness of each multiplex on 20 to 50 single leukocytes of the couple. Each couple had at least one embryo not carrying the risk haplotype, suitable for transfer. The couples with NF1 and achondroplasia both delivered a healthy, unaffected baby. The pregnancy is ongoing in the couple with the EXT2 variant. PGT-M is now easily handled for standard situations, with semiautomated protocols that do not need extensive setups. De novo mutations however present a unique challenge, because of the impossibility in most cases of determining the phase of the disease-causing variant. We present a patient-centric approach with individualized protocols. Limitations, reasons for caution Allele drop-out could lead to misdiagnosis of the embryo. To avoid that, 6 flanking STRs (3 proximal and 3 distal) and genotyping of the variant should be performed. When possible, it is good practice to pre-define the different haplotypes with the parents of the patients. Wider implications of the findings: The increasing number of laboratories offering off-the-shelf testing with NGS panels and semi-automated PGT can fulfil demand for routine situations. However in more complex cases, diagnostic-quality NGS and individualized PGT-M programmes are needed. These cases also remind us that PGT-M requires extensive multidisciplinarity to maximize the chance of successful outcome. Trial registration number Not applicable
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Open Access
May 29, 2024
Fitaqi Almada + 2
