45. A COMPLETE SOLUTION FOR BETA-THALASSEMIA PGT TEST

Abstract

Introduction Couple who are both β-thalassemia carriers usually come to PGT-M test for healthy offsprings. STR-linkage analysis is a well-known solution but Allele Drop-Out, high mutation rate and stutter peaks are still major challenges. Karyomapping is a common technique but it's costly and not be combined with PGT-A test, which may result in multiple biopsy or unaffected but aneuploidy embryos. In this study, we report two cases of β-thalassemia PGT test which uses single nucleotide polymorphisms (SNPs) for linkage analysis. By using specific primers in WGA process, the Allele Drop-out rate decreases to Materials and method Two β-thalassemia-carrier couples underwent IVF procedure, resulted in 11 embryos. Specific primers were desinged to amplify whole HBB-coding region and SNPs within 300kb flanking the HBB gene. Day 5 biopsied samples were used for whole genome amplification with designed specific primers in order to decrease ADO in HBB gene region and SNP markers, using DOPlify kit (RHS). WGA product and specific primers were used for PCR reaction to enrich HBB region and interested SNPs. Mix of WGA and enrichment PCR product was used for libraries preparation using Nextera XT DNA Library Prep Kit (Illumina) and sequenced on Miseq System. Nexus copy number was used for CNV analysis andMiseq Reporter was used for mutation detection and SNP calling. Results For the total of 11 samples, three samples resulted in aneuploidy. HBB mutation detection and SNP calling results showed no ADO in all 11 samples, with 3 affected embryos and 8 unaffected embryos. Conclusions We have successfully designed a procedure which allows to combine PGT-A and PGT-M in one single test. This test is more cost-effective and decreases turn-around time compared with other techniques. ADO rate of HBB region and SNP makers is approximately 0%, which make it easier and more reliablefor ADO confirmation among embryos in the same cycle.