Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro

Abstract

Purpose: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization.

Methods: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping.

Results: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results.

Conclusion: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.

Figure 1

Karyomaps of single blastomeres biopsied from cleavage-stage embryos. Paternal haplotypes are represented in blue/red, and maternal haplotypes are represented in orange/green. Haplotypes inherited by the reference are shown in blue/orange. For a detailed description of how the karyomaps are displayed, see the Materials and Methods section. (a) Karyomaps for chromosome 11 in three embryos from a preimplantation genetic diagnosis (PGD) case for β-thalassemia and the unaffected child born following transfer of Embryo 1. The sibling used as a reference to phase the SNP calls is a carrier of the affected paternal allele (blue/orange), whereas Embryo 1 and the child born following the transfer of Embryo 1 are unaffected (red/orange), Embryo 2 is affected (blue/green), and Embryo 3 is a carrier of the paternal allele (blue/orange). Note the consistent pattern of key and non-key SNPs (colored dots) above and below the predicted haploblocks. In addition, note the crossovers in both the paternal (upper) and maternal (lower) chromosomes, common to all samples, indicating crossovers in the reference (boxed). (b) A detailed view of the β-globin locus (HBB) in Embryo 3, including the 2-Mb flanking regions proximal and distal to the gene (gray shading). Note that there are two isolated key SNPs (red dots), presumed miscalls, immediately distal to the gene. (c) Detailed karyomap of the huntingtin locus (HTT), with 2-Mb flanking regions to the left and right, in two single cells from a cleavage-stage embryo in a PGD case for Huntington disease. Note the recombination of the paternal chromosome (top) in the 2-Mb 3′-flanking region. The location of this recombination upstream of the gene is fixed by the presence of three non-key SNPs (in Cell 1) and by one key SNP and three non-key SNPs (in Cell 2), supporting the presence of the same haplotype as the reference. SNP, single-nucleotide polymorphism.

Figure 2

Detailed karyomaps of chromosome 19, in the terminal p13.3 region, for five embryos from a preimplantation genetic diagnosis case for Peutz–Jeghers syndrome, caused by a mutation in the STK11 gene. Also shown is the outcome of conventional testing with two proximal semi-informative STR markers (D19S565 and D19S247) and direct mutation detection. Paternal haplotypes are represented in blue/red, and maternal haplotypes are represented in orange/green. Haplotypes inherited by the reference are shown in blue/orange. The affected child has inherited the mutation in STK11 from the father. Therefore, the reference haplotype (blue) represents the affected haplotype in this case. Note that there is a common crossover on the paternal chromosome between the two STR markers, which indicates that the affected child used as a reference for linkage had a crossover in this position. Furthermore, there are three additional crossovers in the region on the paternal chromosomes in these five embryos and the maternal chromosome 19 is not present in two embryos (haploblock bar grayed out). This complex pattern of crossovers and aneuploidy detected by karyomapping is completely concordant with the STR alleles (table below) and the presence or absence of the mutation (indicated by the + or − in the STR table). However, Embryos 2, 3, and 4 have identical STR results, and only direct mutation testing identifies Embryo 3 as affected. STR, short tandem repeat.

Figure 3

Comparison of targeted haplotyping and direct mutation detection with karyomapping in a nonconcordant single-blastomere sample (Embryo 6) from a preimplantation genetic diagnosis cycle for Crigler–Najjar syndrome, type 1, caused by a mutation in UGT1A1. Paternal haplotypes are represented in blue/red, and maternal haplotypes are represented in orange/green. Haplotypes inherited by the reference are shown in blue/orange. The father and mother are carriers for this autosomal recessive disorder, and the reference child is affected. This means that both the blue and the orange haplotypes carry the mutation. Embryo 6 has the blue paternal and green maternal haplotypes, indicating a paternal carrier. On the basis of the four flanking STR markers and two intragenic markers, along with the mutation analysis (lower panel), this embryo was diagnosed as an unaffected maternal carrier. In the mutation analysis, + indicates the presence of the mutation, and − indicates its absence. Only the intragenic SNP marker identifies the presence of the normal paternal allele. All other markers are ambiguous and were labeled as suspected ADOs. Phasing of the UGT1A1 locus by karyomapping is unequivocal (upper panel) and indicates that the embryo is an unaffected paternal carrier. Note that the terminal regions of both chromosome arms have the characteristic karyomapping patterns of key and non-key informative SNPs, indicating regions (marked by square braces) in which both parents have a chromosome with an identical SNP genotype, i.e., identical by descent. ADO, allele dropout; SNP, single-nucleotide polymorphism; STR, short tandem repeat.