47. PGT-M IN FAMILIES CARRYING "DE NOVO" MUTATION BY COMBINATION OF EMBRYO SANGER SEQUENCING AND KARYOMAPPING

• Published in: Reproductive biomedicine online 2019-08, Vol.39, p.e55-e56
• Main Authors: Becvarova, V., Soldatova, I., Linhartova, E., Trkova, M., Sekowska, M., Diblik, J., Koudova, M., Horacek, J., Bittoova, M., Stejskal, D.
• Format: Article
• Language: English
• Subjects: de novo mutation; karyomapping; monogenic disease
• ISSN: 1472-6483; 1472-6491
• DOI: 10.1016/j.rbmo.2019.04.100

Karyomapping based on genome - wide linkage analysis and using an unique combination of "SNPs (Single Nucleotide Polymorphism) of interest" in each chromosomal region enables to compare patterns carried by parents and other family member of known genetic status (carrier of a possible pathogenic mutation) in an affected region. In case of "de novo" mutation carried by one of the parents (and detected by Sanger sequencing), there is no other family member available for a linkage analysis. The only opportunity to get reference is to perform an acquired embryonal DNA sequencig. DNA amplification from trophectoderm samples was performed using the MDA (multiple displacement amplification) protocol recommended in the Karyomapping methodology. Direct detection of the causal mutations was performed on DNA amplification from trophectoderm by Sanger´s method of gene sequencing. Prior to the embryo analysis, the carrier mutation was verified. WGA products were sequenced and compared with reference sequence of the tested gene. A minimum of 3 embryos are required for the "de novo" mutation testing. Short - read fragments were used for Sanger seguencing. For the indirect genome - wide linkage analysis the Human Karyomap-12v1 bead chip (Illumina) and BlueFuseMulti were used. Four unrelated families were examined by Karyomapping PGT Method. All monogenic diseases were autosomal dominant with known mutation in definite gene (SALL4 - OMIM 607343; FBN2 - 612570; LMNA - 150330; EXT2 - 608210). In all four cases we were able to find an embryo as reference by Sanger sequencing for a conclusive result. The average number of examined embryos was 5.5 (2 - 12) per family. With exception of 1 case (EXT2 family) we always found at least 1 embryo for an embryotransfer. In the FBN2 - family the heart beat pregnancy is reported. The examination scheme of this family is presented in detail. Karyomapping is an applicable method to PGT-M also in families with detected "de novo" mutation. Under the assumption of a known mutation and a sufficient number of embryos as well as a coverage check in the region of interest it is possible to reveal the mutation using the sequenced embryo as a reference family member.