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

Abstract

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 marker-based 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.