Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Arch Med Res. 2016 May;47(4):293-8. doi: 10.1016/j.arcmed.2016.07.006.
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.
地中海贫血症可通过骨髓移植治愈；然而，寻找具有特定 HLA 组合的合适供体仍然是一个主要挑战。通过胚胎植入前遗传学诊断（PGD）预先选择 HLA 系统的脐带血细胞对于解决 HLA 匹配骨髓供体稀缺的问题非常有用。
本研究纳入了一对携带地中海贫血特征的夫妇和一名受影响的孩子。我们使用 HLA 和β珠蛋白基因座上的信息性 STR 标记物开发了一种单细胞多重荧光 PCR 方案。该方案在从夫妇外周血中分离的单个淋巴细胞上进行了广泛优化。将优化后的方案应用于夫妇第 3 天 IVF 胚胎卵裂期 IV 期活检的单个卵裂球。
对第 3 天活检的四个 IVF 胚胎和每个胚胎的单个卵裂球进行了联合β地中海贫血突变和 HLA 分型的遗传诊断。其中一个胚胎被诊断为纯合正常的地中海贫血突变，与现有受影响的同胞 HLA 匹配。
优化后的方案在 PGD 临床周期中运行良好，可选择具有所需 HLA 系统的无地中海贫血症胚胎。
