C32 Comprehensive microarray genetic analyses on single cell(s) from polar bodies or embryos to determine 23-chromosome aneuploidy, structural chromosome aberrations and genome-wide scans using single nucleotide polymorphisms (SNPs); to identify what partner provided the extra chromosome in aneuploid embryos; and to determine what embryo implanted following an in vitro fertilization (IVF) cycle

Abstract

not available at time of publication. C32 Comprehensive microarray genetic analyses on single cell(s) from polar bodies or embryos to determine 23-chromosome aneuploidy, structural chromosome aberrations and genome-wide scans using single nucleotide polymorphisms (SNPs); to identify what partner provided the extra chromosome in aneuploid embryos; and to determine what embryo implanted following an in vitro fertilization (IVF) cycle W.G. Kearns1, R. Pen1, A. Benner1, A. Siegel1, E. Widra2, R. Leach1. 1Shady Grove Center for Preimplantation Genetics, 2Shady Grove Fertility Reproductive Science Center, Rockville, Maryland, USA *E-mail: Kearnsw@LabCorp.com (W.G. Kearns) Objective: To amplify DNA from a polar body, a single embryonic blastomere or trophectoderm cell(s) and to perform complex genetic analyses. Design: Prospective study. Materials and Methods: A modified whole genome amplification (WGA) protocol was performed on 513 single cells (112 WBCs, 367 human 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. Two-channel intensity values from the microarray data files were used to create high-resolution copynumber profiles to identify copy number variations (CNVs) for each cell. Data was analyzed with deCODE genetics Disease Miner Professional® and Illumina BeadStudio software. Results: Analyses of blastomeres (n = 367) from 61 day-3 preimplantation embryos and cell lines (n = 34) showed in many cases, a genomic coverage >98%, a heterozygous allele detection rate >90% and a microarray detection rate and genotype call rate >90%. In some cases, call rates exceeded 99%. A 23-chromosome molecular karyotype was obtained from all 367 blastomeres (>8400 individual chromosomes) and all 34 cell lines (>780 individual chromosomes). Structural chromosome imbalances were identified from all 9 cytogenetically abnormal cell lines. Some of these structural chromosome imbalances included: del(8q), add(17p), del(17p), add(4q), add(9p), add(14q), dup(18p), dic(5), del(12p) and del(9p). Genotype information was also obtained for ~370K SNPs for each cell analyzed. These genome wide scans identified disease risks in embryos for prostate cancer, glaucoma, certain cardiovascular conditions and estrogen responsive breast cancer. Our SNP scans also identified 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 can also be identified. Our microarrays can also identify single gene mutations for diseases such as cystic fibrosis, Marfan syndrome, Retinoblastoma or Neurofibromatosis Type 1. A high-resolution copy-number profile also identified CNVs throughout the genome. 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. Support: EMD Serono