Appropriate whole genome amplification and pathogenic loci detection can improve the accuracy of preimplantation genetic diagnosis for deletional α-thalassemia

Objective To improve the accuracy of preimplantation genetic testing (PGT) in deletional alpha-thalassemia patients. Design Article. Patient(s) fifty-two deletional alpha-thalassemia couples. Intervention(s) Whole genome amplification (WGA), Next-generation sequencing (NGS) and PCR mutation loci detection. Main outcome measures WGA, Single nucleotide polymorphism (SNP) and PCR mutation loci detection results; Analysis of embryo chromosome copy number variation (CNV). Results Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) methods for PGT for deletional alpha-thalassemia. Blastocyst biopsy samples (n = 253) were obtained from 52 deletional alpha-thalassemia couples. The results of the comparison of experimental data between groups MALBAC and MDA are as follows: (i) The average allele drop-out (ADO) rate, MALBAC vs. MDA = 2.27% +/- 3.57% vs. 0.97% +/- 1.4%, P=0.451); (ii) WGA success rate, MALBAC vs. MDA = 98.61% vs. 98.89%, P=0.851; (iii) SNP haplotype success rate, MALBAC vs. MDA = 94.44% vs. 96.68%, P=0.409; (iv) The result of SNP haplotype analysis is consistent with that of Gap-PCR/Sanger sequencing results, MALBAC vs. MDA = 36(36/72, 50%) vs. 151(151/181, 83.43%), P=0; (v) Valid SNP loci, MALBAC vs. MDA = 30 +/- 9 vs. 34 +/- 10, P=0.02; (vi) The mean CV values, MALBAC vs. MDA = 0.12 +/- 0.263 vs. 0.09 +/- 0.40, P=0.916; (vii) The average number of raw reads, MALBAC vs. MDA =3244259 +/- 999124 vs. 3713146 +/- 1028721, P=0; (viii) The coverage of genome (%), MALBAC vs. MDA = 5.02 +/- 1.09 vs. 5.55 +/- 1.49, P=0.008. Conclusions Our findings indicate that MDA is superior to MALBAC for PGT of deletional alpha-thalassemia. Furthermore, SNP haplotype analysis combined with PCR loci detection can improve the accuracy and detection rate of deletional alpha-thalassemia.