Validation of preimplantation genetic testing for monogenic disorders (pgt-m) using targeted next generation sequencing

Preimplantation genetic testing for monogenic disorders (PGT-M) is a genetic test that can detect genetic alterations associated with specific single gene disorders in a trophectoderm (TE) biopsy from an IVF embryo. However, the reliability of PGT-M is compromised by both allele dropout (ADO) and amplification failure (AF). The aim of this study was to evaluate the reliability of PGT-M using targeted next generation sequencing (tNGS) in samples of multiple cells and validate this method in TE biopsies. Phase I - Reliability: To demonstrate that a single TE biopsy of 4-5 or more cells is sufficient for PGT-M, fibroblasts were used to prepare 1, 2, 3, 4, or 5 cell samples. Twenty heterozygous single nucleotide polymorphisms (SNP) were tested using tNGS. Phase II – TE validation: Embryos from patients that had undergone PGT-M with clinically established Taqman qPCR genotyping were analyzed. Sequencing primers were designed for 12 different variants, including single nucleotide alterations, deletions, and insertions, and then validated on parental genomic DNA. Linkage primers were also included. Additional primers were designed to target SNPs in which parents had opposite homozygous alleles. Abnormal embryos donated for research were thawed, re-biopsied, and processed with tNGS-based PGT-M while simultaneously being screened for aneuploidy (PGT-A). Phase I: The one-cell samples exhibited a 20% AF (142/720) and 6% ADO (42/720) rate. The 2-cell samples had a 6% AF (42/720) and 2% ADO (12/720) rate. However, the 3-cell, 4-cell, and 5-cell samples exhibited 0% AF and 0% ADO. The PGT-A call rates were 61% for 1-cell samples, 92% for 2-cell samples, 97% for 3-cell samples, and 100% for 4 or 5-cell samples. The median absolute pairwise difference (MAPD) decreased with the increased cell number. Phase II: One or two TE biopsies of 23 embryos from 8 families, tested for 1 to 3 of the 12 variants (MLH1 c.199G>A, RB1 c.1589A>G, SLC16A2 c.604G>A, GNPTAB c.1123C>T, GNPTAB c.3314A>G, WFS1 c.1391C>G, USH2A c.10073G>A, USH2A del exons 5-11, F5 c.1601G>A, GAA c.693-1G>C, GAA c.875A>G, and HEXA c.1274_1277dupTATC), were 100% concordant with previous PGT-M results based on Taqman qPCR genotyping. All the linkage markers showed expected genotype. All the additional expected heterozygous SNPs showed 0% AF and 0% ADO in the TE samples unless aneuploidy caused loss or gain of the SNP on that chromosome. PGT-A analysis of multiple biopsies from the same embryo demonstrated consistent karyotypes. This study demonstrates that a tNGS-based platform can reliably produce PGT-M diagnoses with extremely low AF and ADO when a TE biopsy consists of 3 or more cells.