Haitao Wu1,2, Xiaoting Shen2, Lei Huang3,4, Yanhong Zeng2, Yumei Gao5, Lin Shao5, Baomin Lu2, Yiping Zhong2, Benyu Miao2, Yanwen Xu2, Yali Wang2, Yubin Li2, Luoxing Xiong6,7,8, Sijia Lu5, X Sunney Xie3,6,7, Canquan Zhou9. 1. Reproductive Medicine Center, Jiangmen Central Hospital, Affiliated Jiangmen Hospital of Sun Yat-Sen University, Jiangmen, Guangdong, 529030, China. 2. Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, China. 3. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, 01238, USA. 4. Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Boston, MA, 02115, USA. 5. Yikon Genomics Co., Ltd., 1698 Wangyuan Road, Building #26, Fengxian District, Shanghai, 201400, China. 6. Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing, 100871, China. 7. Beijing Advanced Innovation Center for Genomics, Peking University, Beijing, 100871, China. 8. Peking-Tsinghua Center for Life Sciences (CLS), Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China. 9. Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong, 510080, China. zhoucanquan@hotmail.com.
Abstract
PURPOSE: This paper aims to investigate the feasibility of performing pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS) simultaneously by a universal strategy without the requirement of genotyping relevant affected family members or lengthy preliminary work on linkage analysis. METHODS: By utilizing a universal Mutated Allele Revealed by Sequencing with Aneuploidy and Linkage Analyses (MARSALA) strategy based on low depth whole genome sequencing (~3x), not involving specific primers' design nor the enrichment of SNP markers for haplotype construction. Single-sperm cells and trephectoderm cells from in vitro fertilized embryos from a couple carrying HBB mutations were genotyped. Haplotypes of paternal alleles were constructed and investigated in embryos, and the chromosome copy number profiles were simultaneously analyzed. RESULTS: The universal MARSALA strategy allows the selection of a euploid embryo free of disease mutations for in uterus transfer and successful pregnancy. A follow-up amniocentesis was performed at 17 weeks of gestation to confirm the PGD/PGS results. CONCLUSION: We present the first successful PGD procedure based on genotyping multiple single-sperm cells to obtain SNP linkage information. Our improved PGD/PGS procedure does not require genotyping the proband or relevant family members and therefore can be applicable to a wider population of patients when conducting PGD for monogenic disorders.
PURPOSE: This paper aims to investigate the feasibility of performing pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS) simultaneously by a universal strategy without the requirement of genotyping relevant affected family members or lengthy preliminary work on linkage analysis. METHODS: By utilizing a universal Mutated Allele Revealed by Sequencing with Aneuploidy and Linkage Analyses (MARSALA) strategy based on low depth whole genome sequencing (~3x), not involving specific primers' design nor the enrichment of SNP markers for haplotype construction. Single-sperm cells and trephectoderm cells from in vitro fertilized embryos from a couple carrying HBB mutations were genotyped. Haplotypes of paternal alleles were constructed and investigated in embryos, and the chromosome copy number profiles were simultaneously analyzed. RESULTS: The universal MARSALA strategy allows the selection of a euploid embryo free of disease mutations for in uterus transfer and successful pregnancy. A follow-up amniocentesis was performed at 17 weeks of gestation to confirm the PGD/PGS results. CONCLUSION: We present the first successful PGD procedure based on genotyping multiple single-sperm cells to obtain SNP linkage information. Our improved PGD/PGS procedure does not require genotyping the proband or relevant family members and therefore can be applicable to a wider population of patients when conducting PGD for monogenic disorders.
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