Validation of single nucleotide polymorphism (SNP) microarray PGD on single cell(s) from embryos

Abstract

OBJECTIVE: To amplify DNA from a single blastomere and to perform complex SNP microarray genetic analyses. Validation studies will be discussed.DESIGN: Prospective study.MATERIALS AND METHODS: Multiple displacement amplification was performed on 962 single cells from 270 cytogenetically abnormal embryos as determined by 10-probe FISH and 34 known cell lines. Invariant DNA genomic loci were used to ensure the entire genome was amplified and TaqMan PCR to ensure heterozygous allele amplification. The Illumina Human HapMap or Cyto-12 microarrays were employed including genotype data for 300-370K SNPs. Data was analyzed with deCODE Genetics Disease Miner Professional and Illumina GenomeStudio and KaryoStudio software.RESULTS: Analyses of blastomeres and 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%. A 23-chromosome molecular karyotype was obtained from over 99% of all blastomeres and all 34 cell lines. Structural chromosome imbalances were identified from all cytogenetically abnormal cell lines. These aberrations included deletions and duplications. Genotype information was also obtained for over 85% of all SNPs analyzed. Upon reconstruction of all cells from individual embryos, 5% of embryos showed aneuploidy for the same 3 chromosomes, 64% of embryos were mosaic diploid/aneuploid, 23% were mosaic aneuploid and 8% were complex mosaics. These genome wide scans can also identifiy subtle DNA aberrations associated with disorders such as Beckwith-Wiedemann syndrome, some types of Prader Willi/Angelman syndrome, DiGeorge syndrome 1/ Velocardiofacial syndrome and some forms of autism. Uniparental disomy and Copy Number Variants can also be identified.CONCLUSION: We successfully validated and obtained complex genetic information from 962 single embryonic cells using a modified WGA protocol and SNP microarray analyses. These analyses may be performed on polar bodies, blastomeres or trophectoderm cells. OBJECTIVE: To amplify DNA from a single blastomere and to perform complex SNP microarray genetic analyses. Validation studies will be discussed. DESIGN: Prospective study. MATERIALS AND METHODS: Multiple displacement amplification was performed on 962 single cells from 270 cytogenetically abnormal embryos as determined by 10-probe FISH and 34 known cell lines. Invariant DNA genomic loci were used to ensure the entire genome was amplified and TaqMan PCR to ensure heterozygous allele amplification. The Illumina Human HapMap or Cyto-12 microarrays were employed including genotype data for 300-370K SNPs. Data was analyzed with deCODE Genetics Disease Miner Professional and Illumina GenomeStudio and KaryoStudio software. RESULTS: Analyses of blastomeres and 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%. A 23-chromosome molecular karyotype was obtained from over 99% of all blastomeres and all 34 cell lines. Structural chromosome imbalances were identified from all cytogenetically abnormal cell lines. These aberrations included deletions and duplications. Genotype information was also obtained for over 85% of all SNPs analyzed. Upon reconstruction of all cells from individual embryos, 5% of embryos showed aneuploidy for the same 3 chromosomes, 64% of embryos were mosaic diploid/aneuploid, 23% were mosaic aneuploid and 8% were complex mosaics. These genome wide scans can also identifiy subtle DNA aberrations associated with disorders such as Beckwith-Wiedemann syndrome, some types of Prader Willi/Angelman syndrome, DiGeorge syndrome 1/ Velocardiofacial syndrome and some forms of autism. Uniparental disomy and Copy Number Variants can also be identified. CONCLUSION: We successfully validated and obtained complex genetic information from 962 single embryonic cells using a modified WGA protocol and SNP microarray analyses. These analyses may be performed on polar bodies, blastomeres or trophectoderm cells.