65. SUCCESSFUL STRATEGY OF COMPREHENSIVE PRE-IMPLANTATION GENETIC TESTING FOR BETA-THALASSEMIA-HEMOGLOBIN E DISEASE AND CHROMOSOME BALANCE USING KARYOMAPPING

Abstract

Introduction Thalassemia syndrome and hemoglobinopathy are the world commonest monogenic disease and cause health and economic burden worldwide. Pre-implantation genetic testing (PGT) is an alternative to traditional prenatal diagnosis (PND). However, the nature of a vast variety of mutations makes molecular genetic testing sophisticated and labor intensive. Modern haplotyping using SNP array (aSNP) and Karyomapping algorithm would omit molecular analysis development process and provide chromosome balance information at the same time. This study applied Karyomapping for PGT-M of beta-thalassemia-hemoglobin E (Hb E) disease in 2 clinical PGT cycles in comparison to PCR testing techniques. Material and methods: Two families at risk of having beta-thalassemia-Hb E disesase offspring decided to join the project following thoroughly counselling and inform consent was obtained. The patients underwent routine IVF procedures. Embryo biopsy was performed on Day-5 post-fertilization and the biopsied trophectoderm underwent whole genome amplification. SNP array with Karyomapping analysis was carried out for haplotyping as well as copy number variation (CNV). Multiplex PCR with mini-sequencing was performed alongside for confirmation standard molecular mutation analysis. Informative polymorphic marker was also included for contamination identification Results Thirteen and nine embryos with good morphology from families YW and CS were chosen for PGT, respectively. Karyomapping results of family YW (beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease) revealed four normal, two beta-thalassemia trait, one Hb E trait and six affected with beta-thalassemia-Hb E disease. Standard mutation analysis using multiplex fluorescent PCR and mini-sequencing confirmed haplotyping results in all embryos. In addition, Karyomapping demonstrated three embryos with chromosome unbalanced, i.e. 45,XY, -19 (affected), 47,XY, +16 (beta-thalassemia trait) and 47,XX, +21 (beta-thalassemia trait). Therefore, four normal (three male and one female) and one Hb E trait embryos were fulfilled for transfer. One normal male embryo was chosen for transfer and one normal male baby was delivered. Prenatal and postnatal DNA testing confirmed PGT results. Karyomapping results of family CS (beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease) revealed six Hb E trait and three affected with beta-thalassemia-Hb E disease. Standard mutation analysis using multiplex fluorescent PCR and mini-sequencing confirmed haplotyping results in all embryos. Additionally, Karyomapping demonstrated two embryos with chromosome unbalanced, i.e. 45,XY, -21 (affected) and 45,XY, -22 (Hb E trait). Therefore, five Hb E trait embryos were fulfilled for transfer. One Hb E trait embryo was chosen for transfer and one ongoing pregnancy was resulted. Polymorphic marker analysis revealed the absence of extraneous DNA contamination. Conclusions Two clinical PGT-M cycles using Karyomapping were performed for two families at risk of having beta–thalassemia (c.17A>T)-Hb E (c.26G>A) disease offspring. Additional standard multiplex PCR with mini-sequencing analysis confirmed haplotyping results of Karyomapping. However, aSNP provides the benefit of extra information of chromosome balance. This study demonstrated that kryomapping can omit the risk of transfer chromosomally unbalanced embryos, termination of abnormal chromosome pregnancy later and the birth of abnormal chromosome babies. Therefore, Karyomapping provides an accurate, quick, time saving for protocol development, universal PGT-M method for every monogenic disease, and also the advantage of CNV information which is common in pre-implantation embryos.