HLA-G molecule plays an important role on immune response regulation and has been implicated on the inhibition of T and natural killer cell cytolytic function and inhibition of allogeneic T-cell proliferation. Due to its immune-modulator properties, the HLA-G gene expression has been associated with the outcome of allograft and of autoimmune, infectious, and malignant disorders. Several lines of evidence indicate that HLA-G polymorphisms at the 5'-upstream regulatory region (5' URR) and 3'-untranslated region (3' UTR) may influence the HLA-G expression levels. Because Brazilians represent one of the most heterogeneous populations in the world with the widest HLA-G coding region variability already detected among the studied populations, a high level of variability and haplotype diversity would be expected in Brazilians. On this basis, the 5' URR, coding, and 3' UTR variability were evaluated in a Brazilian series consisting of 100 healthy bone marrow donors, as well as the linkage disequilibrium pattern along the gene and the extended haplotypes encompassing several gene segment variations. The HLA-G locus seems to present six different HLA-G lineages showing functional variations mainly in nucleotides of the regulatory regions. Differences were observed at the 5' URR in positions that either coincide with or are close to transcription factor-binding sites and at the 3' UTR mainly in positions that have already been reported to influence HLA-G mRNA availability. We report several lines of evidence for balancing selection acting on the regulatory regions, which may indicate that these HLA-G lineages may be related to the differential HLA-G expression profiles.

HLA-G has a relevant role in immune response regulation. The overall structure of the HLA-G coding region has been maintained during the evolution process, in which most of its variable sites are synonymous mutations or coincide with introns, preserving major functional HLA-G properties. The HLA-G promoter region is different from the classical class I promoters, mainly because (i) it lacks regulatory responsive elements for IFN-γ and NF-κB, (ii) the proximal promoter region (within 200 bases from the first translated ATG) does not mediate transactivation by the principal HLA class I transactivation mechanisms, and (iii) the presence of identified alternative regulatory elements (heat shock, progesterone and hypoxia-responsive elements) and unidentified responsive elements for IL-10, glucocorticoids, and other transcription factors is evident. At least three variable sites in the 3′ untranslated region have been studied that may influence HLA-G expression by modifying mRNA stability or microRNA binding sites, including the 14-base pair insertion/deletion, +3142C/G and +3187A/G polymorphisms. Other polymorphic sites have been described, but there are no functional studies on them. The HLA-G coding region polymorphisms might influence isoform production and at least two null alleles with premature stop codons have been described. We reviewed the structure of the HLA-G promoter region and its implication in transcriptional gene control, the structure of the HLA-G 3′UTR and the major actors of the posttranscriptional gene control, and, finally, the presence of regulatory elements in the coding region.

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The HLA-G molecule presents immunomodulatory properties that might inhibit immune responses when interacting with specific Natural Killer and T cell receptors, such as KIR2DL4, ILT2 and ILT4. Thus, HLA-G might influence the outcome of situations in which fine immune system modulation is required, such as autoimmune diseases, transplants, cancer and pregnancy. The majority of the studies regarding the HLA-G gene variability so far was restricted to a specific gene segment (i.e., promoter, coding or 3' untranslated region), and was performed by using Sanger sequencing and probabilistic models to infer haplotypes. Here we propose a massively parallel sequencing (NGS) with a bioinformatics strategy to evaluate the entire HLA-G regulatory and coding segments, with haplotypes inferred relying more on the straightforward haplotyping capabilities of NGS, and less on probabilistic models. Then, HLA-G variability was surveyed in two admixed population samples of distinct geographical regions and demographic backgrounds, Cyprus and Brazil. Most haplotypes (promoters, coding, 3'UTR and extended ones) were detected both in Brazil and Cyprus and were identical to the ones already described by probabilistic models, indicating that these haplotypes are quite old and may be present worldwide.