Genome sequencing of human in vitro fertilisation embryos for pathogenic variation screening

Abstract

Whole-genome sequencing of preimplantation human embryos to detect and screen for genetic diseases is a technically challenging extension to preconception screening. combining preconception genetic screening with preimplantation testing of human embryos facilitates the detection of de novo mutations and self-validates transmitted variant detection in both the reproductive couple and the embryo's samples. Here we describe a trio testing workflow that involves whole-genome sequencing of amplified DNA from biopsied embryo trophectoderm cells and genomic DNA from both parents. Variant prediction software and annotation databases were used to assess variants of unknown significance and previously not described de novo variants in five single-gene preimplantation genetic testing couples and eleven of their embryos. Pathogenic variation, tandem repeat, copy number and structural variations were examined against variant calls for compound heterozygosity and predicted disease status was ascertained. Multiple trio testing showed complete concordance with known variants ascertained by single-nucleotide polymorphism array and uncovered de novo and transmitted pathogenic variants. this pilot study describes a method of whole-genome sequencing and analysis for embryo selection in high-risk couples to prevent early life fatal genetic conditions that adversely affect the quality of life of the individual and families. Whole-genome sequencing in the iVf clinic. For over two decades, preimplantation genetic testing (PGT) has been available for couples who are aware they carry a genetic condition or have had a child affected by a genetic disease. In vitro fertilisation (IVF) used in conjunction with monogenic PGT is available for couples to prevent transmission of known hereditary monogenic disorders. PGT for aneuploidy screens embryos for large segmental or whole-chromosome copy number changes and is commonly used for older women (>35 years) who have a history of infertility, miscarriages or chromosomally abnormal conceptions 1-3. The most recent developments in clinical PGT are low-coverage next-generation sequencing and Karyomapping, which uses a highly polymorphic single-nucleotide polymorphism (SNP) microarray to identify disease-causing haplotypes. Nextgeneration sequencing PGT for aneuploidy (typically <0.1× depth) is useful for high-throughput screening at a reasonable cost for detecting chromosomal aneuploidies, structural variations and large copy-number variations (CNVs) 4-6. In addition to pedigree analysis for monogenic disorders, Karyomapping has been reported to identify partial chromosomal aneuploidies as small as 1.8 Mb 7. For couples seeking to ascertain their risk of having an affected child, around 6,000 diseases exist that may be genetically screened for 8. A mutation or disease-causing variant in one or both copies of approximately 5,000 human genes can cause a syndromic disease or phenotype 9-14. Between 0.5-5% of infants are born with a genetic condition or disorder 15,16. The preconception genetic screening...

Genome-Wide Disease Screening in Early Human Embryos with Primary Template-Directed Amplification

“…This is due to the inherent limitations of currently used WGA methods that introduce method-specific artifacts, limiting the accuracy of downstream analyses for a given WGA method. Previous studies have performed whole genome sequencing of embryo biopsies using multiple displacement amplification (MDA) 29,30 . However, those studies were also hampered by the artifacts introduced by MDA, including loss of coverage and uneven coverage that hamper variant calling.…”

Mysteries and unsolved problems of mammalian fertilization and related topics

“…Will it be possible to assess genomic status of spermatozoa without “killing” them? Methods proposed by Watanabe et al [ 316 ] and Yang et al [ 317 ] are very labor-intensive and time-consuming; therefore, conventional genomic analyses of cells from preimplantation embryos [ 318 , 319 ] are still the best way today to avoid the birth of offspring with serious developmental problems.…”

Whole-genome risk prediction of common diseases in human preimplantation embryos

“…These approaches rely on the use of a PRS 7 that combines the effects of tens or hundreds or thousands of genetic variants into a single predictor. However, due to the limited quantity and quality of DNA in single-cell or few-cell embryo biopsies, attempts to comprehensively profile the genomes of embryos are costly and time intensive 8 – 10 , suffer from inaccuracies related to allele dropout, require extended relatives 2 or rely on imputation, which hampers the detection of rare deleterious variants in genes like BRCA1 11 . To overcome these limitations, we have extended a strategy 1 for whole-genome reconstruction (WGR) that uses parental genome sequencing and embryo genotyping to predict the inherited genome sequence of an embryo.…”