Novel Multiplex Fluorescent PCR-Based Method for HLA Typing and Preimplantational Genetic Diagnosis of β-Thalassemia.
Thalassemia is curable by bone marrow transplantation; however, finding suitable donors with defined HLA combination remains a major challenge. Cord blood stem cells with preselected HLA system through preimplantation genetic diagnosis (PGD) proved very useful for resolving scarce HLA-matched bone marrow donors.



A thalassemia trait couple with an affected child was included in this study. We used informative STR markers at the HLA and beta globin loci to develop a single cell multiplex fluorescent PCR protocol. The protocol was extensively optimized on single lymphocytes isolated from the couple's peripheral blood. The optimized protocol was applied on single blastomeres biopsied from day 3 cleavage stage IVF embryos of the couple.



Four IVF embryos biopsied on day 3 and a single blastomere of each were provided for genetic diagnosis of combined β-thalassemia mutations and HLA typing. Of these, one embryo was diagnosed as homozygous normal for the thalassemia mutation and HLA matched with the existing affected sibling.



The optimized protocol worked well in PGD clinical cycle for selection of thalassemia-unaffected embryos with the desired HLA system.
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Sharifeh
S
Khosravi
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Mansour
M
Salehi
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Mahboobeh
M
Ramezanzadeh
Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Hamed
H
Mirzaei
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran; Acquired Immunodeficiency Research Center, Isfahan University of Medical Sciences, Isfahan, Iran; Pediatric Inherited Diseases Research Center, Isfahan University of Medical Sciences, Isfahan, Iran; Gerfa Namayesh Azmayesh (GENAZMA) Science and Research Institute, Isfahan, Iran. Electronic address: r_salehi@med.mui.ac.ir.
Rasoul
R
Salehi
D005260:Female; D005307:Fertilization in Vitro; D005453:Fluorescence; D056426:Genetic Loci; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D055544:beta-Globins / Q000235:genetics; D017086:beta-Thalassemia / Q000235:genetics / Q000276:immunology*
D055544:beta-Globins
HLA typing; Multiplex fluorescent PCR; Preimplantation genetic diagnosis; STR markers; β-thalassemia