Abstract
The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230–232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 homozygous EBV B cell line resulted in one HLA-B*38:68Q/B*38:01:01:01 heterozygous and one HLA-B*38:68Q homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of HLA-B*38:01:01:01 in homozygous cells was 2.2 fold higher than HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the expression of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 2.0 fold higher than HLA-B*38:68Q homozygous cells. The HLA-B*38:68Q expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the HLA-B*38:01:01:01 homozygous cells was 1.5 fold higher than that of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 1.6 fold higher than that of HLA-B*38:68Q homozygous cells. The treatment of HLA-B*38:68Q homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that HLA-B*38:68Q is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function.
Highlights
(Q) Human Leukocyte Antigen (HLA)-B*38:68Q allele, which carries a 9-nucleotide deletion at codon 230–232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas[9] gene editing technology
We identified a new HLA B allele that is similar to HLA-B*38:01:01:01 except for a nine-nucleotide deletion (5′-CTTGTGGAG-3′) at codon 230 to 232 that results in a coding shift at α3 domain of HLA-B38 (Fig. 1A)
We successfully introduced gene editing in 84% of clones and achieved precise deletion at codon 230–232 at exon 4 in 5 alleles
Summary
(Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230–232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas[9] gene editing technology. In allogeneic hematopoietic stem cell transplantation (HSCT), HLA mismatches between donor and recipient leads to strong alloimmune responses that result in either graft versus host disease (GVHD) or engraftment failure[4,5,6]. Mutations such as insertions, deletions in HLA sequences can lead to the introduction of stop codons and/. Failure to identify HLA null alleles in donors may result in HLA mismatches that can stimulate allogeneic T cells and trigger GVHD in HSCT7. It is important to determine the expression patterns of abnormally expressed HLA variants[7]
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