title: Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses.
abstract: In vitro fertilization (IVF), preimplantation genetic diagnosis (PGD), and preimplantation genetic screening (PGS) help patients to select embryos free of monogenic diseases and aneuploidy (chromosome abnormality). Next-generation sequencing (NGS) methods, while experiencing a rapid cost reduction, have improved the precision of PGD/PGS. However, the precision of PGD has been limited by the false-positive and false-negative single-nucleotide variations (SNVs), which are not acceptable in IVF and can be circumvented by linkage analyses, such as short tandem repeats or karyomapping. It is noteworthy that existing methods of detecting SNV/copy number variation (CNV) and linkage analysis often require separate procedures for the same embryo. Here we report an NGS-based PGD/PGS procedure that can simultaneously detect a single-gene disorder and aneuploidy and is capable of linkage analysis in a cost-effective way. This method, called "mutated allele revealed by sequencing with aneuploidy and linkage analyses" (MARSALA), involves multiple annealing and looping-based amplification cycles (MALBAC) for single-cell whole-genome amplification. Aneuploidy is determined by CNVs, whereas SNVs associated with the monogenic diseases are detected by PCR amplification of the MALBAC product. The false-positive and -negative SNVs are avoided by an NGS-based linkage analysis. Two healthy babies, free of the monogenic diseases of their parents, were born after such embryo selection. The monogenic diseases originated from a single base mutation on the autosome and the X-chromosome of the disease-carrying father and mother, respectively.
authors.affiliation: Department of Obstetrics and Gynecology, Third Hospital, Peking University, Beijing 100191, China;
authors.forename: Liying
authors.initials: L
authors.lastname: Yan
authors.affiliation: Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing 100871, China;
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.affiliation: Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing 100871, China;
authors.forename: Liya
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Obstetrics and Gynecology, Third Hospital, Peking University, Beijing 100191, China; Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China;
authors.forename: Jin
authors.initials: J
authors.lastname: Huang
authors.affiliation: Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing 100871, China;
authors.forename: Fei
authors.initials: F
authors.lastname: Ma
authors.affiliation: Department of Obstetrics and Gynecology, Third Hospital, Peking University, Beijing 100191, China; Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China;
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing 100871, China;
authors.forename: Yaqiong
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing 100871, China;
authors.forename: Mingshan
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Obstetrics and Gynecology, Third Hospital, Peking University, Beijing 100191, China;
authors.forename: Ying
authors.initials: Y
authors.lastname: Lian
authors.affiliation: Department of Obstetrics and Gynecology, Third Hospital, Peking University, Beijing 100191, China;
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: Department of Obstetrics and Gynecology, Third Hospital, Peking University, Beijing 100191, China; Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China;
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.affiliation: Yikon Genomics, Inc., Taizhou, Jiangsu, 225300, China;
authors.forename: Sijia
authors.initials: S
authors.lastname: Lu
authors.affiliation: Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing 100871, China; Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing 100871, China; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; tangfuchou@pku.edu.cn jie.qiao@263.net xie@chemistry.harvard.edu.
authors.forename: Fuchou
authors.initials: F
authors.lastname: Tang
authors.affiliation: Department of Obstetrics and Gynecology, Third Hospital, Peking University, Beijing 100191, China; Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China; Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; tangfuchou@pku.edu.cn jie.qiao@263.net xie@chemistry.harvard.edu.
authors.forename: Jie
authors.initials: J
authors.lastname: Qiao
authors.affiliation: Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing 100871, China; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138 tangfuchou@pku.edu.cn jie.qiao@263.net xie@chemistry.harvard.edu.
authors.forename: X Sunney
authors.initials: XS
authors.lastname: Xie
mesh_terms: D000328:Adult; D000782:Aneuploidy; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002869:Chromosome Aberrations; D025063:Chromosome Disorders / Q000175:diagnosis / Q000235:genetics; D056915:DNA Copy Number Variations; D005260:Female; D005307:Fertilization in Vitro; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; D050498:Live Birth; D008297:Male; D009154:Mutation; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
keywords: IVF; MALBAC; PGD; chromosome abnormality; monogenic diseases