HLA-F displays highly divergent and frequent haplotype lineages associated with different mRNA expression levels.

HLA-F is one of the most conserved loci among the HLA gene family. The exact function of HLA-F is still under investigation. HLA-F might present tolerogenic features, participate in the stabilization of HLA molecules in open conformation, and also participate in the recycling of HLA molecules. Here we evaluate the variability and haplotype structure of the HLA-F distal promoter segment (from -1893 to -943) and how this segment is correlated with the coding region. Variability at the promoter segment was surveyed in 196 Brazilian samples using second-generation sequencing. The HLA-F promoter region presents two major haplotype lineages. Most of the variable sites are in perfect linkage and associated with a single promoter haplotype, here named F∗distal-C. This haplotype is associated with F∗01:01:02 alleles, while alleles from the F∗01:01:01 or F∗01:03 groups present closely related promoter sequences. F∗distal-C is quite frequent in Brazil and in worldwide populations, with frequencies ranging from 8.41% at the Iberian Population in Spain to 34.34% in Vietnam. F∗distal-C is also present in Neanderthal and Denisovan samples. In silico analyses demonstrated that F∗distal-C presents a different transcription factor binding profile compared with other HLA-F promoters. Moreover, individuals carrying this haplotype present higher HLA-F mRNA expression levels. Functional studies are required to define the exact mechanism underlying this higher HLA-F mRNA expression level associated with F∗distal-C and F∗01:01:02 alleles.

Hla-mapper: An application to optimize the mapping of HLA sequences produced by massively parallel sequencing procedures.

A challenging task when more than one HLA gene is evaluated together by second-generation sequencing is to achieve a reliable read mapping. The polymorphic and repetitive nature of HLA genes might bias the read mapping process, usually underestimating variability at very polymorphic segments, or overestimating variability at some segments. To overcome this issue we developed hla-mapper, which takes into account HLA sequences derived from the IPD-IMGT/HLA database and unpublished HLA sequences to apply a scoring system. This comprehends the evaluation of each read pair, addressing them to the most likely HLA gene they were derived from. Hla-mapper provides a reliable map of HLA sequences, allowing accurate downstream analysis such as variant calling, haplotype inference, and allele typing. Moreover, hla-mapper supports whole genome, exome, and targeted sequencing data. To assess the software performance in comparison with traditional mapping algorithms, we used three different simulated datasets to compare the results obtained with hla-mapper, BWA MEM, and Bowtie2. Overall, hla-mapper presented a superior performance, mainly for the classical HLA class I genes, minimizing wrong mapping and cross-mapping that are typically observed when using BWA MEM or Bowtie2 with a single reference genome.

HLA-E regulatory and coding region variability and haplotypes in a Brazilian population sample.

The HLA-E gene is characterized by low but wide expression on different tissues. HLA-E is considered a conserved gene, being one of the least polymorphic class I HLA genes. The HLA-E molecule interacts with Natural Killer cell receptors and T lymphocytes receptors, and might activate or inhibit immune responses depending on the peptide associated with HLA-E and with which receptors HLA-E interacts to. Variable sites within the HLA-E regulatory and coding segments may influence the gene function by modifying its expression pattern or encoded molecule, thus, influencing its interaction with receptors and the peptide. Here we propose an approach to evaluate the gene structure, haplotype pattern and the complete HLA-E variability, including regulatory (promoter and 3'UTR) and coding segments (with introns), by using massively parallel sequencing. We investigated the variability of 420 samples from a very admixed population such as Brazilians by using this approach. Considering a segment of about 7kb, 63 variable sites were detected, arranged into 75 extended haplotypes. We detected 37 different promoter sequences (but few frequent ones), 27 different coding sequences (15 representing new HLA-E alleles) and 12 haplotypes at the 3'UTR segment, two of them presenting a summed frequency of 90%. Despite the number of coding alleles, they encode mainly two different full-length molecules, known as E*01:01 and E*01:03, which corresponds to about 90% of all. In addition, differently from what has been previously observed for other non classical HLA genes, the relationship among the HLA-E promoter, coding and 3'UTR haplotypes is not straightforward because the same promoter and 3'UTR haplotypes were many times associated with different HLA-E coding haplotypes. This data reinforces the presence of only two main full-length HLA-E molecules encoded by the many HLA-E alleles detected in our population sample. In addition, this data does indicate that the distal HLA-E promoter is by far the most variable segment. Further analyses involving the binding of transcription factors and non-coding RNAs, as well as the HLA-E expression in different tissues, are necessary to evaluate whether these variable sites at regulatory segments (or even at the coding sequence) may influence the gene expression profile.

HLA-G variability and haplotypes detected by massively parallel sequencing procedures in the geographicaly distinct population samples of Brazil and Cyprus.

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.

HLA-E coding and 3' untranslated region variability determined by next-generation sequencing in two West-African population samples.

HLA-E is a non-classical Human Leucocyte Antigen class I gene with immunomodulatory properties. Whereas HLA-E expression usually occurs at low levels, it is widely distributed amongst human tissues, has the ability to bind self and non-self antigens and to interact with NK cells and T lymphocytes, being important for immunosurveillance and also for fighting against infections. HLA-E is usually the most conserved locus among all class I genes. However, most of the previous studies evaluating HLA-E variability sequenced only a few exons or genotyped known polymorphisms. Here we report a strategy to evaluate HLA-E variability by next-generation sequencing (NGS) that might be used to other HLA loci and present the HLA-E haplotype diversity considering the segment encoding the entire HLA-E mRNA (including 5'UTR, introns and the 3'UTR) in two African population samples, Susu from Guinea-Conakry and Lobi from Burkina Faso. Our results indicate that (a) the HLA-E gene is indeed conserved, encoding mainly two different protein molecules; (b) Africans do present several unknown HLA-E alleles presenting synonymous mutations; (c) the HLA-E 3'UTR is quite polymorphic and (d) haplotypes in the HLA-E 3'UTR are in close association with HLA-E coding alleles. NGS has proved to be an important tool on data generation for future studies evaluating variability in non-classical MHC genes.

Insights into HLA-G Genetics Provided by Worldwide Haplotype Diversity.

Human leukocyte antigen G (HLA-G) belongs to the family of non-classical HLA class I genes, located within the major histocompatibility complex (MHC). HLA-G has been the target of most recent research regarding the function of class I non-classical genes. The main features that distinguish HLA-G from classical class I genes are (a) limited protein variability, (b) alternative splicing generating several membrane bound and soluble isoforms, (c) short cytoplasmic tail, (d) modulation of immune response (immune tolerance), and (e) restricted expression to certain tissues. In the present work, we describe the HLA-G gene structure and address the HLA-G variability and haplotype diversity among several populations around the world, considering each of its major segments [promoter, coding, and 3' untranslated region (UTR)]. For this purpose, we developed a pipeline to reevaluate the 1000Genomes data and recover miscalled or missing genotypes and haplotypes. It became clear that the overall structure of the HLA-G molecule has been maintained during the evolutionary process and that most of the variation sites found in the HLA-G coding region are either coding synonymous or intronic mutations. In addition, only a few frequent and divergent extended haplotypes are found when the promoter, coding, and 3'UTRs are evaluated together. The divergence is particularly evident for the regulatory regions. The population comparisons confirmed that most of the HLA-G variability has originated before human dispersion from Africa and that the allele and haplotype frequencies have probably been shaped by strong selective pressures.