O-270 Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing
Authors: Janssen A , Koeck R , Essers R , Van Dijk W , Drüsedau M , Meekels J , Yaldiz B , Van de Vorst M , Dreesen J , De Die-Smulders C , Coonen E , Brunner H , Van den Wijngaard A , Paulussen A , Zamani Esteki M 
Published: July 2024

Study question

Can a simple, and scalable assay be developed to concurrently perform all forms of preimplantation genetic testing (PGT), effectively addressing the increasing demand?

Summary answer

Whole genome sequencing-based haplarithmisis offers a single-assay solution for all forms of PGT, enhancing resolution and diagnosis in complex cases.

What is known already

State-of-the-art preimplantation genetic testing (PGT) methods, including genotyping-by-sequencing (GBS)-based haplotyping, enable PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A) in a single assay. However, they are labour intensive, only partially cover the genome, require a close relative to determine meiotic and mitotic origin of aneuploidies, and are troublesome for consanguineous families and in complex genomic contexts. Furthermore, the prevailing PGT approach for mitochondrial DNA disorders (PGT-MT) involves a PCR-based method. This underscores the need to develop a universal PGT method that streamlines laboratory protocols and provides genome-wide coverage to address genetic disorders, even in complex genomic loci.

Study design, size, duration

In a retrospective pilot study, we compared whole genome sequencing (WGS) at 30-40X with GBS (6 embryos, 2 families, 3 indications). We carried out subsampling of WGS data and determined 10X as the optimal diagnostic depth-of-coverage. We clinically validated our approach by comparing WGS to GBS for PGT-M and PGT-SR (31 embryos, 16 families, 22 indications), to shallow sequencing for PGT-SR (10 embryos, 3 families) and to PCR-based PGT for PGT-MT (10 embryos, 2 families).

Participants/materials, setting, methods

DNA from parents and close relative(s) and whole genome amplified (WGAed) DNA from embryos, underwent WGS with subsequent haplarithmisis. Quality metrics, such as Mendelian inconsistency rates, number of informative SNPs, and haplotype concordance were assessed. Translocation breakpoints were identified utilizing aberrantly aligned paired sequencing reads. Additionally, single-nucleotide variants (SNVs), including mitochondrial DNA SNVs, were detected by allele counting. Finally, segregational origin was determined via a parents-only haplarithmisis approach that uses the embryo as phasing reference.

Main results and the role of chance

WGS-PGT alleviates technical limitations by decreasing WGA artifacts by 68.4%, increasing breadth of coverage by 4-fold, and decreasing wet-lab turnaround time by 2.5-fold. Owing to its inherent higher resolution and improved genomic coverage, 5 out of 13 – previously inconclusive – embryos with genetic disorders in complex genomic regions could be assigned a diagnosis. Furthermore, we show the ability to directly detect single- and few-base pair genetic variants in any region of interest. Importantly, we show trio-based PGT-A for aneuploidy origin, an approach we term PGT-AO that – using the parents-only genotype calls – can detect the segregational origin of aneuploidies without the need of a close relative as reference for phasing. The segregational origin detected by parents-only haplarithmisis was concordant with the segregational origin determined from haplarithmisis when using a close relative. Unbalanced translocations could be detected indirectly via haplotyping or directly at single base resolution using a structural variant caller. Mitochondrial heteroplasmy level calculation from WGS yielded comparable results to PCR-based methods, both in blastomere and trophectoderm biopsy samples.

Limitations, reasons for caution

Direct variant detection with an average coverage of 10X may occasionally be inadequate, as certain locations may lack sufficient coverage (&lt;5 reads). Inherently to the limited sample size of PGT-MT families, WGS-PGT needs further validation. The need for invasive trophectoderm biopsies of preimplantation embryos remains the key limitation of WGS-PGT.

Wider implications of the findings

Our approach enables all forms of PGT in a single assay. Direct variant detection complements haplarithmisis, addressing inconclusive findings and aiding families with a de novo variant or lacking a close relative for phasing. Finally, PGT-AO facilitates research on the origin of aneuploidies in preimplantation embryos and their clinical outcome.

Trial registration number

not applicable