Pushing the limits of preimplantation genetic diagnosis (PGD): a Karyomapping and Next Generation Sequencing (NGS) stress test

Abstract

We sought to examine the limits of embryo genetic analysis by performing PGD and aneuploidy screening (PGS) in 1) a consanguineous mating for, 2) two disease-causing point mutations and 3) HLA-genotyping to match 3 affected children while 4) detecting genetic recombination and potential maternal cell contamination, and 5) 24-chromosome aneuploidy screening with NGS. Karyomapping utilizes ∼300,000 Single Nucleotide Polymorphisms (SNPs) across the human genome and allows high-resolution analysis for nearly every gene. We used karyomapping to rapidly design a PGD test targeting the HBB gene and the highly recombinant HLA locus to identify embryos with one or both healthy HBB alleles and an HLA genotype matching the children in need of transplantation. Cheek-swab kits were used to collect DNA samples from the parents and affected children. Mutation status of each individual was confirmed by direct sequencing. DNA samples were analyzed using the Infinium HumanKaryomap-12 DNA Analysis Kit from Illumina. BluFuse Multi software was used to compare the SNP profiles of each family member to identify informative SNPs, regions of consanguinity, and determine HLA phase for each affected child. NGS was used for 24-chromosome aneuploidy screening. We identified sufficient informative SNPs surrounding the HBB gene and HLA region to track the inheritance of familial haplotypes. As expected, the same HBB haplotypes were observed in each affected child. Two children were found to have the same set of HLA haplotypes, while the third inherited the opposite paternal HLA haplotype. This is consistent with the HLA-typing reported by an external laboratory. Recombination and consanguineous regions were observed but did not impact our ability to determine genetic phase. We successfully used karyomapping to build a complex PGD test, which would have been highly challenging using previous methods. Moreover, we determined the HLA phases of all three children simultaneously and confirmed that recombination and consanguinity would not interfere with diagnosis or HLA matching. Our success addresses concerns about using karyomapping for HLA matching and diagnosing embryos of consanguineous families. Karyomapping combined with PGS by Next-Generation Sequencing represents a great advance to the field, 25 years after its inception.