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

Abstract

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‐A promoter, coding, and 3′UTR sequences in a Brazilian cohort, and their evolutionary aspects

“…Consequently, variability in these segments might be underestimated. Moreover, variability in the HLA‐A promoter region has not been well characterized so far …”

Next‐generation sequencing of HLA‐G based on long‐range polymerase chain reaction

“…Here we present a long‐range PCR sequencing technique for genotyping HLA‐G using the Ion Torrent platform. Castelli et al have previously published an NGS method for the MiSeq platform to sequence the HLA‐G , HLA‐E , and HLA‐F genes . Apart from the fact that the sequencing platform differs between the two methods, we have also used different library preparation methods.…”

“…Early in vitro studies have described that MNAse have a preference of cleaving at specific motifs . However, even with commercially available fragmentation kits, specifically designed for NGS library preparation, fragmentation bias is observed in GC‐rich regions …”

The role of HLA‐G in parasitic diseases

“…These 56 HLA‐G alleles are organized in 20 allele groups that encode 2 truncated ( G*01:05 N and G*01:13 N ) and 18 full‐length protein variants. A few studies have thoroughly explored the genetic structure and patterns of diversity of the regulatory and coding regions of HLA‐G in worldwide human populations, including Brazil, Cyprus and 14 populations from the 1000 Genomes Project, using both Sanger and next‐generation sequencing methodologies . In spite of the low sequencing coverage, the dataset from the 1000 Genomes Project revealed the existence of at least 35, 81 and 17 variation sites in the 5′ URR (upstream regulatory region), coding and 3′ UTR portions of HLA‐G , respectively.…”

“…Signature of balancing selection has been disclosed at both flanking regulatory regions . Notwithstanding, due to the strong linkage disequilibrium across the entire HLA‐G gene, thus encompassing the 5′ URR, coding and 3′ UTR segments, it is not possible to unequivocally establish the actual targets of balancing selection and if any segment merely reflects a hitchhiking effect . Even so, balancing selection may result in the maintenance of different HLA‐G lineages, associated with different expression patterns over time .…”