SNP array-based copy number and genotype analyses for preimplantation genetic diagnosis of human unbalanced translocations

Abstract

Preimplantation genetic diagnosis (PGD) for chromosomal rearrangements (CR) is mainly based on fluorescence in situ hybridisation (FISH). Application of this technique is limited by the number of available fluorochromes, the extensive preclinical work-up and technical and interpretative artefacts. We aimed to develop a universal, off-the-shelf protocol for PGD by combining single-nucleotide polymorphism (SNP) array-derived copy number (CN) determination and genotyping for detection of unbalanced translocations in cleavage-stage embryos. A total of 36 cleavage-stage embryos that were diagnosed as unbalanced by initial PGD FISH analysis were dissociated (n=146) and amplified by multiple displacement amplification (MDA). SNP CNs and genotypes were determined using SNP array. Epstein-Barr Virus-transformed cell lines with known CR were used for optimising the genomic smoothing (GS) length setting to increase signal to noise ratio. SNP CN analysis showed 23 embryos (64%) that were unbalanced in all blastomeres for the chromosomes involved in the translocation, 5 embryos (14%) that were normal or balanced in all blastomeres and 8 embryos (22%) that were mosaic. SNP genotyping, based on analysis of informative SNP loci with opposing homozygous parental genotypes, confirmed partial monosomies associated with inheritance of unbalanced translocation in surplus embryos. We have developed a universal MDA-SNP array technique for chromosome CN analysis in single blastomeres. SNP genotyping could confirm partial monosomies. This combination of techniques showed improved diagnostic specificity compared with FISH and may provide more reliable PGD analysis associated with higher embryo transfer rate.

Figure 1

(a) Influence of GS settings on analytic specificity, that is, mean % of CN=2 call (diploid) in normal EBV-LCLs. (single cells n=14, multi cells n=10, g-DNA samples n=2). The complete genome (represented by approximately 262 000 SNP probes) was analysed. (b) Influence of GS settings on analytical resolution, that is, % of correct CN calling from specific regions with known aberrations, that is, diploid region 3p26.1 → 3qtel (CN=2) and partial monosomy 3p26.3 → 3p26.1 (CN=1) in EBV-LCL 16310 (single cells n=5, multi cells n=1, g-DNA sample n=1).

Figure 2

Single-cell CN analyses using the MDA-SNP array. An example of a single blastomere of case 2-1, embryo 1 is shown. Upper panel for both chromosomes shows the log2ratio of blastomere over reference signals (top: chromosome 5, bottom: chromosome 7). The lower panel for both chromosomes (top: chromosome 5, bottom: chromosome 7) shows the CN segments (‘CN_segments') derived thereof (gains are indicated as blue blocks, losses are indicated as red blocks). The color reproduction of this figure is available at the European Journal of Human Genetics online.

Figure 3

Genotype analysis showing parental contributions to the genotype of case 2–1, embryo 1, for the two centric segments (5pter → 5q33 and 7pter → 7q22) and the two translocation segments (5q33 → 5qter and 7q22 → 7qter). The analysis was based on informative parental loci, that is, opposing homozygous genotype calls in the couple. Blue bars indicate paternal-only contribution, without maternal contribution to the embryo's genotype and green bars indicate maternal-only contribution. Note that for the hemizygous region (7q22 → 7qter) associated with unbalanced translocation in the embryo, there is preferential genetic contribution of one parental allele only, that is, the allele of the partner of the translocation carrier (in this case maternal-only contribution). The color reproduction of this figure is available at the European Journal of Human Genetics online.