The FCGR3A gene encodes for the receptor important for antibody-dependent natural killer cell-mediated cytotoxicity. FCGR3A gene polymorphisms could affect the success of monoclonal antibody therapy. Although polymorphisms, such as the FcγRIIIA-V158F and -48L/R/H, have been studied extensively, an overview of other polymorphisms within this gene is lacking. To provide an overview of FCGR3A polymorphisms, we analysed the 1000 Genomes project database and found a total of 234 polymorphisms within the FCGR3A gene, of which 69%, 16%, and 15% occur in the intron, UTR, and exon regions respectively. Additionally, only 16% of all polymorphisms had a minor allele frequency (MAF) > 0.01. To facilitate (full-length) analysis of FCGR3A gene polymorphism, we developed a FCGR3A gene-specific amplification and sequencing protocol for Sanger sequencing and MinION (Nanopore Technologies). First, we used the Sanger sequencing protocol to study the presence of the V158F polymorphism in 76 individuals resulting in frequencies of 38% homozygous T/T, 7% homozygous G/G and 55% heterozygous. Next, we performed a pilot with both Sanger sequencing and MinION based sequencing of 14 DNA samples which showed a good concordance between Sanger- and MinION sequencing. Additionally, we detected 13 SNPs listed in the 1000 Genome Project, from which 11 had MAF > 0.01, and 10 SNPs were not listed in 1000 Genome Project. In summary, we demonstrated that FCGR3A gene is more polymorphic than previously described. As most novel polymorphisms are located in non-coding regions, their functional relevance needs to be studied in future functional studies.
HLA‐DRA encodes the alpha chain of the HLA‐DR protein, one of the classical HLA class II molecules. Reported polymorphism within HLA‐DRA is currently limited compared with other HLA genes, as only a single polymorphism encodes an amino acid difference in the translated protein. Since this SNP (rs7192, HLA00662.1:g.4276G>T p.Val217Leu) lies within exon 4, in the region encoding the cytoplasmic tail, the resulting protein is effectively monomorphic. For this reason, in‐depth studies on HLA‐DRA and its function have been limited. However, analysis of sequences from the 1000 Genomes Project and preliminary data from our lab reveals unrepresented polymorphism within HLA‐DRA, suggesting a more complex role within the MHC than previously assumed. This study focuses on elucidating the extent of HLA‐DRA polymorphism, and extending our understanding of the gene's role in HLA‐DR~HLA‐DQ haplotypes. Ninety‐eight samples were sequenced for full‐length HLA‐DRA, and from this analysis, we identified 20 novel SNP positions in the intronic sequences within the 5711 bp region represented in IPD‐IMGT/HLA. This polymorphism gives rise to at least 22 novel HLA‐DRA alleles, and the patterns of intronic and 3′ UTR polymorphism correspond to HLA‐DRA~HLA‐DRB345~HLA‐DRB1~HLA‐DQB1 haplotypes. The current understanding of the organization of the genes within the HLA‐DR region assumes a single lineage for the HLA‐DRA gene, as opposed to multiple gene lineages, such as in HLA‐DRB. This study suggests that the intron and 3′ UTR polymorphism of HLA‐DRA indicates different lineages, and represents the HLA‐DRA~HLA‐DRB345~HLA‐DRB1~HLA‐DQB1 haplotypes.
Our study demonstrates that the nucleotide variability of the HLA-E gene is much higher than previously known, albeit in only a limited number of individuals. Overall only 2 variants, HLA-E*01:01 and *01:03, are frequently present worldwide, suggesting that balancing selection is acting on HLA-E.
Natural killer (NK) cells are innate lymphocytes that can kill diseased- or virally-infected cells, mediate antibody dependent cytotoxicity and produce type I immune-associated cytokines upon activation. NK cells also contribute to the allo-immune response upon kidney transplantation either by promoting allograft rejection through lysis of cells of the transplanted organ or by promoting alloreactive T cells. In addition, they protect against viral infections upon transplantation which may be especially relevant in patients receiving high dose immune suppression. NK cell activation is tightly regulated through the integrated balance of signaling via inhibitory- and activating receptors. HLA class I molecules are critical regulators of NK cell activation through the interaction with inhibitory- as well as activating NK cell receptors, hence, HLA molecules act as critical immune checkpoints for NK cells. In the current review, we evaluate how NK cell alloreactivity and anti-viral immunity are regulated by NK cell receptors belonging to the KIR family and interacting with classical HLA class I molecules, or by NKG2A/C and LILRB1/KIR2DL4 engaging non-classical HLA-E or -G. In addition, we provide an overview of the methods to determine genetic variation in these receptors and their HLA ligands.
The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles.
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