Comprehensive SNP microarray genetic analyses on single cell(s) from polar bodies or embryos to determine 23-chromosome aneuploidy, structural chromosome aberrations and complex genetic disorders

Abstract

OBJECTIVE: To successfully amplify DNA from a single embryonic cell and to perform complex PGD/PGS genetic testing.DESIGN: Prospective studyMATERIALS AND METHODS: A modified whole genome amplification (WGA) protocol was performed on 565 single blastomeres from 61 embryos and 34 known cell lines. We used invariant DNA genomic loci to ensure the entire genome was amplified and TaqMan PCR to ensure heterozygous allele amplification. The Illumina HumanHap370 microarray was employed to determine chromosome aberrations and genotype data for 370K SNPs. Data was analyzed with deCODE genetics Disease Miner Professionaland Illumina BeadStudio and KaryoStudio software.RESULTS: Analyses of 565 blastomeres (n=61 embryos) and 34 cell lines showed in many cases, a genomic coverage > 98%, a heterozygous allele detection rate > 90% and a microarray detection rate and genotype call rate > 90%. Our DNA amplification protocol permits us to successfully identify ploidy in all cells without having to determine parental DNA genotypes. 69% of the embryos were mosaic diploid/aneuploid, 25% were mosaic aneuploid and 7% were complex mosaics. Structural chromosome imbalances were identified from all cytogenetically abnormal cell lines. Genotype information was also obtained for 370K SNPs from each cell. These genome wide scans identified complex genetic disorders such as DiGeorge syndrome, some deletion forms of Prader-Willi and Angelman syndrome, uniparental disomy and some single gene disorders. Copy number variations (CNVs) were also identified. Using parental, embryonic and fetal genotypic data, we can also determine which partner provided the extra chromosome in aneuploid embryos and which embryo implanted.CONCLUSIONS: We successfully obtained complex genetic information from single embryonic cells using a modified WGA protocol and microarray analyses. These analyses may be performed on polar bodies, blastomeres or trophectoderm cells. OBJECTIVE: To successfully amplify DNA from a single embryonic cell and to perform complex PGD/PGS genetic testing. DESIGN: Prospective study MATERIALS AND METHODS: A modified whole genome amplification (WGA) protocol was performed on 565 single blastomeres from 61 embryos and 34 known cell lines. We used invariant DNA genomic loci to ensure the entire genome was amplified and TaqMan PCR to ensure heterozygous allele amplification. The Illumina HumanHap370 microarray was employed to determine chromosome aberrations and genotype data for 370K SNPs. Data was analyzed with deCODE genetics Disease Miner Professionaland Illumina BeadStudio and KaryoStudio software. RESULTS: Analyses of 565 blastomeres (n=61 embryos) and 34 cell lines showed in many cases, a genomic coverage > 98%, a heterozygous allele detection rate > 90% and a microarray detection rate and genotype call rate > 90%. Our DNA amplification protocol permits us to successfully identify ploidy in all cells without having to determine parental DNA genotypes. 69% of the embryos were mosaic diploid/aneuploid, 25% were mosaic aneuploid and 7% were complex mosaics. Structural chromosome imbalances were identified from all cytogenetically abnormal cell lines. Genotype information was also obtained for 370K SNPs from each cell. These genome wide scans identified complex genetic disorders such as DiGeorge syndrome, some deletion forms of Prader-Willi and Angelman syndrome, uniparental disomy and some single gene disorders. Copy number variations (CNVs) were also identified. Using parental, embryonic and fetal genotypic data, we can also determine which partner provided the extra chromosome in aneuploid embryos and which embryo implanted. CONCLUSIONS: We successfully obtained complex genetic information from single embryonic cells using a modified WGA protocol and microarray analyses. These analyses may be performed on polar bodies, blastomeres or trophectoderm cells.