VALIDATION OF AN INTEGRATED PGT-A STRATEGY FOR PARALLEL ASSESSMENT OF ANEUPLOIDY, PLOIDY LEVEL, AND PRESENCE OF MAJOR MICRODELETIONS IN A SINGLE TROPHECTODERM BIOPSY

Abstract

To evaluate the diagnostic performance of an expanded PGT platform that includes simultaneous assessment of aneuploidy, ploidy status and the most common pathogenic microdeletions. The diagnostic panel evaluated in this study includes ploidy level and 9 common microdeletion (<10Mb) syndromes [1p36 deletion (1p36, OMIM #607872), Wolf- Hirschhorn (4p16.3, OMIM #194190), Cri-du-Chat (5p15, OMIM #123450), Langer-Giedion (8q23-q24, OMIM #150230), Jacobsen (11q23-q25, OMIM #147791), Prader-Willy/Angelman (15q11.2, OMIM #176270/#105830), Smith-Magenis (17p11.2, OMIM #182290), DiGeorge (22q11.21, OMIM #188400)] selected due to their possible de novo onset, incidence and severity. Validation was performed by sequencing of 395 highly polymorphic Single Nucleotide Polymorphisms (SNPs) across the genome and within critical region of each targeted microdeletion. Detected configurations were evaluated blinded based on B-allelic frequency (BAF) signals and the ratio homozygous/heterozygous calls. Loss of heterozygosity (LOH) (AA/BB only) was interpreted as haploidy or presence of microdeletion. Altered BAF ratios with ranges of 20%-40% (AAB) and 60%-80% (ABB) were interpreted as triploid. Validation of microdeletions was performed on 21 trophoectoderma (TE) biopsies with one or multiple aneuploidies, 3 cell lines and 4 genomic DNA from affected children. Ploidy validation was performed on 21 TE biopsies and 2 cell lines . Ploidy level analysis resulted in 100% concordance per sample (n=23/23; 95%CI:85.7-100.0), considering both cell lines and TE biopsies of previously characterized haploid (n=4),polyploid (n=11) and diploid (n=8) blastocysts. For the 8 microdeletions, concordance rate was 62.5% (1p36: n=5/8), 100% (4p16.3: n=7/7), 60% (5p15: n=3/5), 100% (8q23-q24: n=6/6), 100% (11q23-q25: n=5/5), 100% (15q11.2: n=8/8), 100% (17p11.2: n=3/3), 100% 22q11.21: n=8/8), respectively. No false positives were detected. Interestingly, blinded data analysis confirmed the presence of microdeletions in DNA samples from affected patients. The DiGeorge syndrome, which is the most frequent, has been always correctly classified while critical regions variability within 1p and 5p microdeletions deserve further development. This validation study shows that our simultaneous PGT-A sequencing approach allows an accurate characterization of aneuploidies, ploidy level and microdeletions in the same workflow.