P024 Large scale whole gene sequencing of HLA class i genes reveals locus-specific conservation of intronic sequences

Abstract

Aim We have been sequencing whole gene HLA Class I at large scale at Histogenetics on PacBio RSII® platform since the beginning of July of 2016. To date we have sequenced around 185,000 samples for whole gene Class I. We have found 1645, 1697 and 1664 unique sequences for A, B and C genes, respectively. Most of the intronic sequence information comes from the introns flanking Antigen Recognition Site (ARS) encoding introns. In this study we wanted to analyze all intronic sequences to determine their evolutionary process. Methods We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al. Hum Immunol. 2015 Dec;76(12):963–74). Exons were translated, aligned at the codon level using the ClustalW algorithm in MEGA7 (default settings), and the alignment was back-imposed on the nucleotide sequence. Intronic sequence diversity (d), and exonic nonsynonymous (dN) and synonymous (dS) sequence diversity, were calculated based on all pairwise comparisons of unique alleles using SNPGenie (adaptation of Nei-Gojobori method). Results For HLA-A, dS-exons = 0.0404 while d-introns = 0.0236; for HLA-B, dS-exons = 0.0347 while d-introns = 0.0094; and for HLA-C, dS-exons = 0.0305 while d-introns = 0.0186. Conclusions Our results are consistent with the hypothesis that diversity at synonymous coding sites has been elevated because of linkage to nonsynonymous sites, which experience overdominant (positive balancing) selection. In contrast, intronic sites are less closely linked to nonsynonymous coding sites than are synonymous coding sites, making them more likely to experience recombinational separation from coding sites and subsequent drift, which has acted to homogenize the sequences over evolutionary time thus resulting in locus-specific sequences un the introns.