HLA-F displays highly divergent and frequent haplotype lineages associated with different mRNA expression levels
Introduction
HLA-F is a conserved non-classical HLA class I gene presenting a differentiated expression profile when compared with other HLA class I genes. HLA-F is expressed intracellularly in lymphocytes and monocytes, but it can be detected at the cell surface upon activation [1], [2], [3]. The tissue-specific expression has been reported on tonsil, spleen, thymus [1], trophoblasts [4], [5], and fetal liver [2], [6]. HLA-F expression was also reported for different cell lines, including EBV-transformed lymphoblastoid and monocytic cell lines [7], B, and HUT-78 cell lines [1], J82 transitional cell carcinoma, MOLT-3T cell leukemia, JEG choriocarcinoma, BeWo choriocarcinoma, and 3A-subE SV40 transformed [5]. Moreover, HLA-F expression was detected for gastric, non-small-cell lung and breast cancer [8], [9], [10], and also on esophageal squamous cell carcinoma [11].
Unlike other HLA class I molecules, HLA-F can be exported to the cell surface by a mechanism that is independent of loading with peptides in the endoplasmic reticulum but depends on the HLA-F cytoplasmic tail [12]. HLA-F also presents an intracellular function, associated with other HLA class I molecules at the “open conformer” mode. HLA-F may play a role in the stabilization of open conformers, or even participating in a recycling mechanism of these molecules, possibly being associated with the cross-presentation of exogenous antigens [13], [14]. HLA class I molecules in an “open conformer” manner, including HLA-F, may interact with KIR receptors, thus modulating NK cell function [13], [15], [16]. Moreover, the HLA-F peptide-binding groove is different from those of other class I genes [17]. Because of a mutation (R62W) leading to an open-ended groove that can accommodate long peptides, HLA-F presents peptides of unconventional length [13], [17].
Although its exact function is not well established, HLA-F may present tolerogenic and immunomodulatory properties as other non-classical HLA class I molecules since (a) HLA-F interacts with Natural Killer receptors (NKRs), such as ILT2, ILT4, KIR2DS4, KIR3DL2, KIR3DL1, and KIR3DS1 [1], [13], [15], [16], [17], [18]; (b) HLA-F variants were associated with implantation success during pregnancy [19]; (c) HLA-F is expressed on trophoblasts [4], [5], together with HLA-G, HLA-E and HLA-C [4]; (d) HLA-F molecule is quite conserved at the DNA and protein levels, as also observed for HLA-G and HLA-E [20], [21], [22], [23], [24], [25], [26]; (e) HLA-F is highly conserved among primates, including humans, bonobos, gorillas, orangutans, and chimpanzees, suggesting a critical function in the immune response [25], [27], [28], [29].
Regarding its genetic diversity, there are 31 different HLA-F sequences (or alleles) encoding 6 different full-length proteins described so far at the International Immunogenetics database (IPD-IMGT/HLA, version 3.33.0). However, due to the lack of studies addressing HLA-F variability in worldwide populations, HLA-F might be even more polymorphic. So far, only one study has evaluated HLA-F variability by using massively parallel sequencing in a population-level approach [20], revealing the presence of many new HLA-F alleles. Nevertheless, most of these new alleles encode the same HLA-F molecules already reported, emphasizing that HLA-F is indeed the most conserved HLA class I gene. In addition, there is a lack of studies addressing the HLA-F promoter structure. Variability at the regulatory segments may directly influence the HLA-F expression quantitatively and qualitatively. Here we evaluated the variability and haplotype structure of the HLA-F distal promoter segment in the same Brazilian samples already characterized for the proximal promoter and the coding region [20], correlating the HLA-F haplotypes with mRNA expression levels.
Section snippets
HLA-F distal promoter variability and haplotypes
We evaluated the HLA-F distal promoter variability in 204 individuals from the State of São Paulo, Brazil, associating each promoter haplotype with the coding alleles reported for the same samples in a previous study [20]. All participants gave written informed consent to participate in this study, and the UNESP School of Medicine Ethics Committee (#24157413.7.0000.5411) approved this study protocol. HLA-F amplification and sequencing library preparation was performed as described elsewhere [20]
HLA-F promoter variability and extended haplotypes
We evaluated the HLA-F promoter segment between positions −1893 and −943, considering as +1 the Adenine of the first translated ATG. This comprises a promoter segment of 950 bp, henceforth considered as the HLA-F distal promoter. Within this segment, there were 13 variable sites (Table 1), ten of them presenting alternative alleles with frequencies above 15%, and all fitting Hardy-Weinberg equilibrium expectations (P > 0.05). Nine of these variable sites presented the same allele frequencies,
Discussion
Reports on HLA-F variability are quite scarce, mainly when the promoter segment is included. In fact, as far as we know, a single study evaluated the HLA-F promoter, comprehending approximately 2 kb upstream the first translated ATG, but it only explored a few cell lines [25]. This study described a pattern of variation similar to the one detected in this Brazilian population sample. Moreover, only one study evaluated the complete HLA-F coding segment by second-generation sequencing, but the
Acknowledgements
This work was supported by FAPESP/Brazil (Grant# 2013/17084-2). E.C.C. and C.T.M.J. are supported by CNPq/Brazil (Grants# 302590/2016-1 and 309572/2014-2). R.V.B, J.R., T.H.A.L., and L.C.V.C. are supported by the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES/Brazil).
Conflict of interests
There is no conflict of interest.
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