Development of pre-implantation genetic testing protocol for monogenic disorders (PGT-M) of Hb H disease.

Hb H disease is the most severe form of α-thalassemia compatible with post-natal life. Compound heterozygous α 0 -thalassemia - SEA deletion/α + -thalassemia - 3.7kb deletion is the commonest cause of Hb H disease in Thailand. Preimplantation genetics testing for monogenic disorders (PGT-M) is an alternative for couples at risk of the disorder to begin a pregnancy with a healthy baby. This study aims to develop a novel PCR protocol for PGT-M of Hb H disease - SEA/-3.7kb using multiplex fluorescent PCR. A novel set of primers for α + -thalassemia - 3.7kb deletion was developed and tested. The PCR protocol for α 0 -thalassemia - SEA deletion was combined for Hb H disease - SEA/-3.7kb genotyping. The PCR protocols were applied to genomic DNA extracted from subjects with different thalassemia genotypes and on whole genome amplification (WGA) products from clinical PGT-M cycles of the families at risk of Hb Bart's. The results were compared and discussed. The results showed three PCR products from α + -thalassemia - 3.7kb primer set, and three from α 0 thalassemia SEA primer set. The results were consistent with the known thalassemia genotypes. The novel -α 3.7 primers protocol was also tested on 37 WGA products from clinical PGT-M cycles giving accurate genotyping results and a satisfying amplification efficiency with the ADO rates of 2.7%, 0%, and 0% for HBA2, HBA1, and internal control fragments, respectively. This novel PCR protocol can precisely distinguish Hb H disease - SEA/-3.7kb from other genotypes. Additionally, this is the first PCR protocol for Hb H disease - SEA/-3.7kb which is optimal for PGT-M.