Simultaneous detection of chromosomal aneuploidy and a monogenic disease by next-generation sequencing with linkage analysis

RBMO VOLUME 38 ISSUE S1 2019

SIMULTANEOUS DETECTION OF CHROMOSOMAL ANEUPLOIDY AND A MONOGENIC DISEASE BY NEXT-GENERATION SEQUENCING WITH LINKAGE ANALYSIS

Murat Çetinkaya, MD, PhD, Mehmet Ali Tüfekçi, PhD, Burcu Umay Kara, MSc, Yeşim Kumtepe Çolakoğlu, MSc, Prof. Semra Kahraman, MD 1

Istanbul Memorial Hospital, Assisted Reproductive Technologies and Reproductive Genetics Centre, Piyale Pasa Bulvari, Okmeydani Sisli Istanbul 34385, Turkey

Introduction: Mutations giving rise to single gene disorders (SGD) are currently directly detected in PGD-M using PCR and Sanger sequencing. However, amplification failures, allelic drop-out (ADO), mosaicism and contamination limits PGT precision and diagnostic efficiency. The current approaches for PGT-M generally uses standard panels pre-designed for each disease. The present study applied whole genome amplification (WGA) of biopsied trophectoderm cells and concurrent next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping on an Ion Torrent Personal Genome Machine (PGM) with the aim of customized testing for each family. Material and Methods: Exome sequencing data of a consanguineous family applying for ART (mother, father and child), who give their informed consent, was used to establish the haplotype of the thalassemia gene locus-based on informative SNPs by using the VcfTools and R software. A total of 88 loci containing 116 SNPs located 2 Mb upstream and 2 Mb downstream of the beta globin gene locus (HBB) were selected for NGS-based SNP haplotyping. These loci were then submitted to a primer design website (www.ampliseq.com; Thermo Fisher Scientific). The WGA products of three embryos generated during the

IVF cycle of the couple and which were diagnosed as aneuploid and genomic DNA of the child were used for the Amplicon Library Preparation (using Ion AmpliSeq™ Library kits) for PGT-M and in parallel for PGT-A. To validate the SNP-based haplotype, a STR markers’-based haplotype was also established. Results: The SNP-based haplotypes of the tested embryos and of the child were successfully constructed by NGS with linkage analysis, using GeneHunter v2.15 software. Out of the three embryos tested with family customized PGT-M panel, the first embryo was found to be haploidentical with the child for the HBB locus. The second embryo shared only the paternal allele, whereas the third embryo was carrying the other parental alleles when compared to the child's alleles. These haplotypes were also verified by classical STR markerbased haplotyping. Read depth of amplicons ranged between 70 and 6800. The parallel PGS diagnoses of the three embryos were identical with the original diagnoses of the respective embryos. The variations of SNP call rates and read depths of the trophectoderm samples were correlated with library concentrations. Conclusions: In conclusion, this approach allows for a successful simultaneous detection of chromosomal aneuploidy and a monogenic disease by NGS with linkage analysis. Especially in but not limited to consanguineous families, establishing family-specific gene panels for PGT-M maximizes allelic information with a minimum number of SNPs without the need for direct mutation testing thus lowering the cost of testing per embryo. Keywords: blastocyst, preimplantation genetic diagnosis, monogenic disorder, thalassemia, aneuploidy doi: 10.1016/j.rbmo.2019.03.027

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ANEUPLOIDY CONCORDANCE BETWEEN TROPHECTODERM AND INNER CELL MASS BY NEXTGENERATION SEQUENCINGIN 100 BLASTOCYSTS

Andrea Victor1,2, Manuel Viottia4, Rajiv McCoy3, Alan Brake1, Jack Tyndall1, Alex Murphy1, Laura Lepkowsky1, Christo Zouvesa4, Frank Barnesa4, Darren Griffin2 1

Zouves Fertility Center, Foster City, California, USA School of Biosciences, University of Kent, Canterbury, United Kingdom 3 Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey, USA 4 Zouves Foundation for Reproductive Medicine, Foster City, California, USA 2

Introduction: A number of clinical trials have reported improved IVF outcomes when embryos are previously vetted for chromosomal aneuploidies, and yet a significant percentage of euploid embryos still fail to implant. Conversely, there is mounting evidence of mosaic embryos leading to pregnancies, and there even exist anecdotal reports of aneuploid embryos resulting in healthy births. Critics of PGT- A point out that a TE biopsy might not correctly represent the entire blastocyst. Genetic discordance between TE and ICM could severely affect the clinical outcomes of PGT-A. Furthermore, a blastocyst classified as aneuploidy might in reality contain a euploid ICM, which could lead to discarding potentially viable embryos in IVFclinics. To date, studies addressing the question of concordance between TE and ICM have relied oncurrently superseded technologies and/or small sample sizes. Here we analyze 100 blastocysts that wereclassified as aneuploid by PGT-A, probing the genetic makup of their paired ICMs using high-resolutionNextGen Sequencing (hr-NGS). Materials & methods: We optimized a method to collect ICM biopsies that are free of TE cell contamination, validated by immunofluorescence for lineage markers. Using this technique we isolated ICM biopsies from 100 blastocysts that had previously