The comparison of two whole genome amplification methods: A new method of pre-implantation genetic testing (PGT-M) for Southeast Asian deletion in alpha-thalassemia using high resolution melting analysis

Abstract

Introduction In Southeast Asia, alpha-thalassemia with Southeast Asian (SEA) deletion type is the most common mutations of alpha-thalassemia-1 which can cause severe form known as Bart's hydrops fetalis. For couples which carrier of single gene defects, pre-implantation genetic testing for monogenic (PGT-M) plays a role in genetic testing for selecting of unaffected embryos prior implantation. Recently, high resolution melting (HRM) analysis is a simple, rapid and widely used for genotyping and gene scanning of genetic variation. In this study, we developed HRM analysis method for detection of SEA deletion in alpha-thalassemia and compared two WGA methods, SurePlex DNA Amplification System and REPLI-g Single Cell on the performance of HRM analysis and linkage analysis. Material and Methods Retrospective study was carried out in a single private fertility clinic from 2015 to 2017. The single cell embryos were biopsied on blastocyst stage from couples with disease- carrier of SEA deletion type. The trophectoderm biopsied cells were amplified by two whole genome amplification (WGA), SurePlex DNA Amplification System-PCR based method (Illumina, USA) and REPLI-g MDA Single Cell (QIAGEN, Germany). All WGA products were analyzed with two primer pairs of HRM analysis for detection of SEA deletion type in alpha-thalassemia disease and confirmed with the informative short tandem repeated (STRs) linkage analysis. Results A total of 53 single cell biopsied were amplified with SurePlex-WGA (n=33) and REPLI-g MDA (n=20) which used as template for HRM analysis. The HRM results revealed clearly distinguished between wild type and mutant type of SEA deletion mutation in alpha-thalassemia. Comparing between two WGA methods, the HRM amplification results efficiency and allele drop out (ADO) rate of REPLI- g MDA were 95% (19/20) and 31.57% (6/19), respectively. Meanwhile, for SurePlex-WGA showed 75.76% (25/33) and 28% (7/25), respectively. Furthermore, the HRM melting profile results of REPLI-g MDA showed higher concordance rate with the informative STRs linkage analysis results (63.16%, 12/18) than that of SurePlex-WGA (48%, 12/25). Conclusions HRM analysis is an alternative technique with high sensitivity and accuracy for detecting of alpha-thalassemia mutations. The REPLI-g MDA Single Cell method with HRM analysis is suitable for detection of alpha-thalassemia1 SEA type deletion in PGT-M and provided a good concordance with informative STRs linkage analysis. However, ADO from PCR amplification could be occurred and caused of misdiagnosis which might be affected to the interpretation results of the embryos disease- status. Thus, the mutation analysis method using HRM analysis combined with STRs linkage analysis are recommend in PGT-M to avoid the effected of ADO and increase the accuracy and reliable of the PGT-M results.