Karyomapping—a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan syndrome

Preimplantation genetic diagnosis (PGD) of single gene defects by genetic analysis of single or small numbers of cells biopsied from in vitro fertilization (IVF) embryos is clinically well-established. Targeted haplotyping by multiplex fluorescent polymerase chain reaction (PCR) of closely linked or intragenic short tandem repeat (STR) markers combined with direct mutation detection improves the accuracy of single cell analysis significantly and minimizes potential errors caused by undetected allele dropout (ADO) or contamination [1]. Allele dropout refers to the failure of one of the two alleles of a heterozygous locus to amplify. This makes a heterozygous cell appear homozygous at the affected locus, potentially leading to misdiagnosis. Furthermore, using high order multiplex protocols, this approach has been extended to multiple loci, including analysis of the Human leukocyte antigen (HLA) region for selection of embryos tissue matched to existing sick children and diagnosis of translocation chromosome imbalance [2–4]. However, the development of patient, disease or locus-specific protocols, and testing with single cells, is time-consuming and labour intensive. Also, this targeted approach only provides limited information on chromosome aneuploidy, which is recognized to be a major cause of IVF failure and pregnancy loss. As an alternative, we developed, “Karyomapping”—genome wide parental haplotyping using high density single nucleotide polymorphism (SNP) genotyping. Karyomapping provides a comprehensive method for linkage-based diagnosis of any single gene defect [5]. Genotyping of the parents and a close relative of known disease status, to phase informative SNP loci, eliminates the need for customized test development and, as Karyomapping defines four sets of SNP markers for each of the parental chromosomes, it allows simultaneous high-resolution molecular cytogenetic analysis. Thus, meiotic trisomies, including their parental origin, can be identified by the presence of both haplotypes from one parent in segments of the chromosome, resulting from the inheritance of two chromosomes with different patterns of recombination. Moreover, monosomies or deletions can be identified by the absence of either chromosome haplotype from the parent of origin [5]. Mitotic chromosome duplication, which can arise through malsegregation of chromosomes in the cleavage divisions following fertilization, cannot be detected by Karyomapping per se, since the sequence of both chromosomes is identical. However, chromosome duplications may be clinically less significant, since they are often associated with poor morphology and developmental arrest. In the past we have demonstrated that Karyomapping could be used for the detection of cystic fibrosis status in single cells [5]. Here we provide proof of principle for the widespread clinical application of Karyomapping, first by adapting the protocol for clinical use in a regular PGD timeframe (24 h) and secondly by detection of the autosomal dominant condition Marfan syndrome. Performing Karyomapping “as if in a clinical setting” for confirmation of results of an existing PGD case provides strong evidence of the applicability of Karyomapping and, in this case, led to a twin birth.