Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran; Fetal Health Research Center, Hope Generation Foundation, Tehran, Iran; Gene Clinic, Tehran, Iran.
Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.
Biol Blood Marrow Transplant. 2018 Aug;24(8):1575-1580. doi: 10.1016/j.bbmt.2018.03.024. Epub 2018 Apr 9.
Hematopoietic stem cell transplantation (SCT) is the only therapeutic option in a number of heritable hematologic disorders and hematologic cancers. Many parents and families fail to find an HLA-identical donor for their affected family member. In such cases, conceiving for a "savior baby" remains the only option, especially in countries without access to national registries. By means of next generation sequencing (NGS) techniques, in a single experiment on single-cell products of in vitro fertilization, a healthy HLA-identical embryo can be implanted in the uterus of a concerned mother. The patient can therefore benefit from cord blood SCT along with confirming that the fetuses are not suffering from the heritable disorder. This study is an attempt to study the feasibility of preimplantation HLA sequencing on single blastomeres using NGS. Two couples who had previously undergone preimplantation genetic diagnosis of β-thalassemia and their overall 10 embryos were studied and their 5 HLA loci were typed in high resolution through multiple displacement amplification and NGS of single cells. For 88.9% of the 90 HLA alleles, conclusive HLA typing in 4 digit sets was made. HLA alleles were typed; 1 ambiguity in the allelic group and 4 ambiguities in the protein level were observed that were then unraveled by haplotype analysis. Amplification efficiency was 93.3% with an allele drop-out (ADO) rate of 22.2% (6 alleles dropped from a maximum of 27 possible ADOs). In this study the feasibility of a new method of preimplantation HLA sequencing via combining the state-of-the-art techniques used in single-cell whole genome amplification, preimplantation genetic diagnosis, and high-resolution HLA typing by NGS has been shown. This method can make preimplantation HLA sequencing a practicable technique in families desperate for an HLA-matched donor.
造血干细胞移植（SCT）是许多遗传性血液疾病和血液系统癌症的唯一治疗选择。许多父母和家庭无法为受影响的家庭成员找到 HLA 完全匹配的供体。在这种情况下，孕育“拯救宝宝”仍然是唯一的选择，尤其是在没有国家登记处的国家。通过下一代测序（NGS）技术，在体外受精的单细胞产物的单次实验中，可以将健康的 HLA 完全匹配的胚胎植入有关母亲的子宫。因此，患者可以受益于脐带血 SCT，并确认胎儿没有遗传疾病。本研究旨在尝试使用 NGS 对单细胞进行植入前 HLA 测序的可行性。研究了之前接受过β-地中海贫血症植入前遗传诊断的两对夫妇及其总共 10 个胚胎，并通过多次置换扩增和单细胞 NGS 对其 5 个 HLA 基因座进行了高分辨率分型。90 个 HLA 等位基因中有 88.9%可得出 4 位数字的明确 HLA 分型。HLA 等位基因分型显示，在等位基因组中有 1 个模糊，在蛋白质水平有 4 个模糊，然后通过单倍型分析解决了这些模糊问题。扩增效率为 93.3%，等位基因缺失（ADO）率为 22.2%（从最多 27 个可能的 ADO 中丢失 6 个等位基因）。本研究表明，通过结合单细胞全基因组扩增、植入前遗传诊断和 NGS 高分辨率 HLA 分型等最先进技术，新的植入前 HLA 测序方法具有可行性。对于迫切需要 HLA 匹配供体的家庭来说，这种方法可以使植入前 HLA 测序成为一种可行的技术。
