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Apr 2023, Vol 11, Issue 2
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Original Article
Successful Strategy of Pre-implantation Genetic Testing for Beta-Thalassemia (c.17A>T Mutation)-Hb E Disease Using Multiplex Fluorescent PCR and Mini-Sequencing
Worashorn Lattiwongsakorn1, Natpat Jansaka1, Sirivipa Piyamongkol2, Tawiwan Pantasri1, Theera Tongsong1, Wanwisa Suriya1, Wirawit Piyamongkol1
1Department of Obstetrics and Gynaecology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
2Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand

DOI: 10.15296/ijwhr.2023.xx
Viewed : 249 times
Downloaded : 96 times.

Keywords : Beta-thalassemia-Hb E disease, Embryo selection, Mini-sequencing, Multiplex fluorescent polymerase chain reaction, Pre-implantation genetic testing for monogenic disorders
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Abstract
Objectives: Hemoglobin E disease, c.26G>A variant of beta-globin gene, is the most common hemoglobinopathy in Asia. Compound heterozygotes inheriting Hb E disease and beta-thalassemia generate beta-thalassemia-Hb E disease with severe anemia. This study aimed to develop a pre-implantation genetic testing for monogenic disorders (PGT-M) protocol for beta–thalassemia (c.17A>T mutation)-Hb E disease (c.26G>A mutation) using multiplex fluorescent polymerase chain reaction (PCR) and mini-sequencing.

Materials and Methods: bthalw1 primers were used to amplify a beta-globin gene fragment covering both mutations, i.e. beta–thalassemia (c.17A>T) and Hb E disease. D21S11 microsatellite marker was included for contamination detection. Novel mini-sequencing primers were designed and tested for detection of both mutations.

Results: Pre-clinical work up of the optimized PGT-M protocol using 20 single buccal cells of a heterozygous subject showed 100% amplification efficiency and 0% allele drop out (ADO) rate for both primers. In clinical PGT-M cycle, 15 embryos were subjected to biopsy. The results showed two normal, one heterozygous for beta-thalassemia, six heterozygous for Hb E disease, one affected for beta-thalassemia-Hb E disease and five with ambiguous results. Two normally diagnosed embryos were chosen for transfer, one singleton pregnancy was obtained. A healthy baby boy was resulted. Postnatal testing confirmed PGT results.

Conclusions: Novel PGT-M protocols for beta-thalassemia-Hb E disease using multiplex fluorescent PCR and mini-sequencing were developed and described here. The protocol was applied in a clinical PGT-M cycle and gave rise to one successful pregnancy and consequently a healthy baby boy. Mini-sequencing was proved to be rapid, accurate and cost-effective protocol for PGT-M.

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