Evaluation of the HLA-F variability in Brazil by next generation sequencing

Aim HLA-F is a non-classical HLA class I gene and is distinguished from its classical counterparts by low allelic polymorphism and distinctive expression patterns. The exact function of HLA-F remains unknown. It is believed that HLA-F has a tolerogenic function, based on its low variability and sequence conservation among primates. Currently, there is little information regarding the HLA-F allelic variation among human populations and available studies evaluated only a fraction of the HLA-F gene segment and/or searched for known alleles only. Here we evaluated the HLA-F variability (from −300 to the stop codon at position +2944, including introns) in 72 samples from the Southeastern Brazil, using next generation sequencing procedures. Methods HLA-F was amplified by PCR using generic primers. Sequencing libraries were obtained by using Nextera XT DNA (Illumina) and sequenced at the MiSeq platform (together with other HLA class I genes). Paired-end sequences were filtered and mapped by using a local software named HLA-mapper, which allows a reliable sequence mapping when several HLA genes are sequenced together. This procedure takes into account data from IMGT/HLA and 1000Genomes databases to properly assign each pair of sequences to the correct gene. Genotype calling was performed combining the Genome Analysis Toolkit (GATK) and local applications to get correct genotypes. Missing alleles and haplotypes were inferred by using the PHASE method, however, the known phase between variable sites (obtained by GATK) was considered. Results We found 39 variable sites arranged into 14 haplotypes. Of those, five were identical to a known haplotype described by the IMGT/HLA database, that includes F*01:01:01:01, F*01:01:01:08, F*01:01:03:01, F*01:01:03:03 and F*01:03:01:01, and they represent 75% of all haplotypes. The remaining haplotypes did present one or two nucleotide differences from a known IMGT/HLA haplotype. However, most of them were found at least twice in the present series. In addition, four new variable sites were detected. Conclusion we present here a strategy to evaluate HLA-F by NGS and the results do indicate that it is indeed conserved at the protein level, but the HLA-F worldwide haplotype variability might be higher than the one already described by IMGT/HLA.