Preimplantation High-Resolution HLA Sequencing Using Next Generation Sequencing

Introduction

HLA genes are a group of highly polymorphic genes located in the extended MHC region (7.6 Mb) on the short arm of chromosome 6 with 17,509 different alleles known so far. They comprise 3 classes from which HLA-A (3.1 kb), HLA-B (3.4 kb), and HLA-C (3.4 kb) loci from class I and HLA-DRB1 (3.7 to 4.8 kb) and HLA-DQB1 (3.7-4.1 kb) in class II genes are clinically important in graft-versus-host disease. HLA typing has been mainly used in donor selection for transplantation, especially hematopoietic stem cell transplantation (HSCT), as a therapeutic option in hematologic malignancies, congenital disorders of the hematopoietic system, metabolic disorders, and autoimmune disorders. Molecular HLA typing is technically challenging because of the large and highly polymorphic features of HLA molecules. Moreover, application of routine amplification techniques for HLA typing encounters technical limitations because of the presence of multiple genes in distant positions and homology to other domains. With the advent of long-range PCR techniques in 1990s. however, HLA typing techniques made a move from predominantly serologic methods to more molecular genetic ones. Eventually, highly accurate molecular techniques, especially next generation sequencing (NGS), made their way through donor registries, enabling high-resolution HLA matching in huge populations and reducing rejection rate. In spite of all the progress, many patients are still unable to find an HLA-identical donor in universal registries, especially in the absence of nation-wide registries, and are enforced to receive tissues from partially matched donors and to go through its complications because SCT is their only therapeutic option.
Along with the advancements in HLA typing, in the early 2000s the introduction of whole genome amplification techniques, including multiple displacement amplification made preimplantation genetic testing possible from a single cell with a scant amount of DNA material. Later on, quests emerged for a way to conceive for an HLA-identical sibling as a donor for the suffering children. The need was fulfilled by performing in vitro fertilization and selecting HLA-identical embryos followed by HSCT from cord blood samples and also bone marrow aspiration. When preimplantation genetic diagnosis (PGD)-HLA was successful, confirmatory chorionic villus sampling/amniocentesis for prenatal diagnosis of HLA type and genotype of the fetus in case of hereditary hematologic disorders was still indicated. However, the demand made its way through genetic centers, and many reports of successful HSCTs after preimplantation HLA testing have been published since then. The molecular techniques used in these procedures have mainly been based on indirect HLA typing using short tandem repeat (STR) markers, which pose limitations to preimplantation HLA typing. This study is an attempt to study the feasibility of preimplantation HLA typing using NGS of single blastomeres.

Methods

Patients

To study the feasibility of preimplantation HLA sequencing, DNA samples were selected from 2 consanguineous couples who were carriers of β-thalassemia and had gone through PGD for their thalassemia status at the Avicenna Research Institute during June and July 2015. DNA materials were acquired from remnants of the previous peripheral blood samples of the 2 couples, the biopsy of their embryos, and peripheral blood of 2 HLA-known healthy control subjects (typed previously by Sanger sequence-based typing and serologic methods). Seven embryos (E1 to E7) were included from the first family (F1) and 3 embryos (E1 to E3) from the second family (F2). The couple gave permission to study a part of their embryo DNA samples for the feasibility study. Moreover, the study was approved by the Ethics Committee of Avicenna Research Institute, which gave special permission for studying the embryonic samples.

Clinical PGD

Couples had undergone a standard in vitro fertilization cycle. A number of 2 cells had been biopsied on day 3 cleavage stage embryos using laser-assisted microdissection of the zona pellucida and washed in PBS.

Single-Cell Whole Genome Amplification

Multiple displacement amplification of the whole genome was carried out using the Illustra GenomiPhi V2 DNA amplification kit (GE Healthcare Life Sciences, Piscataway, NJ). Single cells were transferred by mouth-controlled pipeting into .2-mL PCR tubes containing 3 µL sample buffer from the Illustra GenomiPhi V2 DNA amplification kit. A total of 1.5 µL cell lysis solution (.6 M KOH, 10 mM EDTA, and 100 mM DTT) was added to single cells. Cell lysis was carried out for 10 minutes at 30°C, followed by enzyme inactivation using 1.5 µL neutralizing buffer (a 4:1 mixture of 1M Tris-HCl pH 8.0 and 3M HCl). Isothermal amplification proceeded at 30°C for 4 hours based on the manufacturer's protocol Enzyme inactivation was then carried out during a 10-minute incubation at 65°C. The quality of the amplified DNA samples was assessed on 1% agarose gel in 30 mM TBE buffer for 20 minutes at 140 V electrophoresis using the High Range DNA Ladder (Jena Bioscience GmbH, Jena, Germany). DNA concentrations were also quantified with the Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA).

High-Resolution HLA Typing by NGS

The HLA locus-specific sequences were amplified with the NGSgo-AmpX kit (GenDx, Utrecht, Netherlands) according to the manufacturer's protocol, and the resulting locus-specific amplicons were pooled. The kit provides the LongRange PCR Kit (Qiagen, Hilden, Germany) and multiple primers for the 5 HLA loci including HLA-A, -B, -C, -DRB1, and -DQB1. Gel electrophoresis was run to ensure the amplification quality. Fragmentation and end-repair were performed with the NGSgo-LibrX (GenDx). Fragments were ligated to the barcode-labeled X adapter and the ISP-compatible P adapter. Fragments with an average size of 200 bps were selected using AMPure XP Beads (Beckman Coulter, Pasadena, CA). Libraries were pooled in equimolar concentrations, and the final library concentration was measured using the Ion Library TaqMan Quantitation Kit (Life Technologies). Clonal amplification was performed by means of the Ion PGM Template OT2 200 kit (Life Technologies). The Ion PGM sequencing 200 kit was used for sequencing. The Ion 316 Chip Kit v2 (Life Technologies) with a capacity of 3 million reads was manually loaded. The resulting data were analyzed by NGSengine 2.0.0.5095 software (GenDx).

Results

Overall, 16 samples comprising 6 blood samples corresponding to 2 consanguineous couples and 2 healthy control subjects along with 10 embryonic samples were studied. Whole genome amplification of the embryonic DNA samples was performed successfully. The quality of whole genome amplification is displayed as a sample in Figure 1, which is a representative example of whole genome amplification of 6 embryos from F1.