P-566 The first use of high resolution PGT-A in a large clinical cohort to detect nine common microdeletion and microduplication syndromes

Abstract

Study question Can microdeletion and microduplication syndromes be accurately detected during PGT-A, and if so, what may be the expected prevalence in preimplantation embryos? Summary answer A modified PGT-A method leveraging high density SNP analysis can detect microdeletions/duplications and their prevalence may be higher than previously reported. What is known already Recurrent microdeletion/microduplication (del/dup) syndromes represent a group of conserved phenotypes involving intellectual disability, developmental delay and physical dysmorphia that are tightly associated with specific segmental gains and losses. Approximately 200 del/dup syndromes have been characterized with a collective occurrence of 1/1000 to 1/200 live births. While data suggests that embryonic prevalence may be higher, segmental copy number variation <5 megabases is difficult to accurately detect with most PGT-A technologies. Poor resolution often translates to low sensitivity and specificity with high false positive rates. There are currently no preimplantation del/dup screening tests, leaving patients at risk of transferring an affected embryo. Study design, size, duration The primary objective of this study was to evaluate performance of a PGT-A platform incorporating detection of nine del/dup disorders (based on occurrence and severity). A preliminary group of 300 samples with confirmed del/dup disorders was analyzed to determine SNP density and resolution and estimate sensitivity and specificity. A second cohort of 3000 de-identified preimplantation embryos was used to assess the baseline detection rate. A larger retrospective study of 25,000 preimplantation embryos will be reported. Participants/materials, setting, methods Cell lines and de-identified trophectoderm biopsies were used to develop, verify, and test the detection method. Samples were processed using primary template-directed amplification (PTA), prepared for next-generation sequencing and enriched for selected SNP data. A bioinformatics pipeline was applied to identify genome-wide and del/dup region-specific aneuploidy through a combination of copy number variation and allelic balance analysis. Orthogonal testing was used to confirm the presence of a del/dup when feasible. Main results and the role of chance PGT-A results displayed 98% concordance to standard outcomes with short segmental or mosaic differences near del/dup regions accounting for most discrepancies. Sequencing data indicated that the del/dup-enabled PGT-A pipeline generated, on average, 42-205 quality-filtered SNPs per megabase across the nine target regions, resulting in a reporting rate of 96-99% and a del/dup resolution down to 1 mb. In the control group, an initial accuracy estimate for del/dup-specific detection was 97-99% with sensitivity and specificity ranging from 82-96% and 92-99%, respectively. Occurrence rates in the limited clinical cohort varied between 1-2x the reported live birth prevalence. A larger preimplantation cohort is being re-analyzed, leveraging orthogonal multiplex ligation-dependent probe amplification (MLPA) or medium-depth whole genome sequencing to confirm the presence or absence of del/dups. Given the conservative approach to analysis and detection, to avoid false negatives, realized occurrence for individual del/dup disorders likely represents the upper bound of the true occurrence. Larger cohorts with confirmatory testing and/or outcome data will be necessary to concretely define this data. Limitations, reasons for caution The presence and severity of del/dup syndromes can vary significantly depending on expressivity, penetrance, and the specific genes impacted. This method aims to detect del/dup disorders arising from full copy number gains and losses of genomic segments, or a loss of heterozygosity. Parallel etiologies may not be detected. Wider implications of the findings This study confirms the strength and utility of a high-resolution PGT-A method to accurately detect segmental del/dups that underly syndromic disorders. Results indicate an elevated preimplantation occurrence rate above that seen in live birth. Overall, PGT-A with del/dup detection could deliver more complete results to further inform embryo transfer decisions. Trial registration number No