Capturing Differential Allele-Level Expression and Genotypes of All Classical HLA Loci and Haplotypes by a New Capture RNA-Seq Method.

Fumiko Yamamoto,Shunichi Kato,Shingo Suzuki,Atsuko Shigenari,Seiamak Bahram,Akiko Mizutani,Satoko Morishima,Masafumi Tanaka,Marcelo Fernandez-Viña,Yasuo Morishima,Takashi Shiina,Jerzy K Kulski,Makoto Murata,Sayaka Ito,Yoshie Kametani

doi:10.3389/fimmu.2020.00941

Abstract

The highly polymorphic human major histocompatibility complex (MHC) also known as the human leukocyte antigen (HLA) encodes class I and II genes that are the cornerstone of the adaptive immune system. Their unique diversity (>25,000 alleles) might affect the outcome of any transplant, infection, and susceptibility to autoimmune diseases. The recent rapid development of new next-generation sequencing (NGS) methods provides the opportunity to study the influence/correlation of this high level of HLA diversity on allele expression levels in health and disease. Here, we describe the NGS capture RNA-Seq method that we developed for genotyping all 12 classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5) and assessing their allelic imbalance by quantifying their allele RNA levels. This is a target enrichment method where total RNA is converted to a sequencing-ready complementary DNA (cDNA) library and hybridized to a complex pool of RNA-specific HLA biotinylated oligonucleotide capture probes, prior to NGS. This method was applied to 161 peripheral blood mononuclear cells and 48 umbilical cord blood cells of healthy donors. The differential allelic expression of 10 HLA loci (except for HLA-DRA and HLA-DPA1) showed strong significant differences (P < 2.1 × 10−15). The results were corroborated by independent methods. This newly developed NGS method could be applied to a wide range of biological and medical questions including graft rejections and HLA-related diseases.

Highlights

The highly polymorphic human major histocompatibility complex (MHC), known as the human leukocyte antigen (HLA), expresses class I and II molecules that present antigens to the T-cell receptors as part of the adaptive immune response [1,2,3,4]
We describe a newly developed capture RNA-Seq method for enriched next-generation sequencing (NGS), genotyping, and for quantitating RNA levels of all 12 classical HLA loci [HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 (HLA-DRB3/DRB4/DRB5)] and alleles using over 200 RNA samples isolated from the peripheral blood mononuclear cells (PBMCs, n = 161) and umbilical cord bloods (UCBs, n = 48) of healthy donors
This sample of genotyped Japanese donors represented more than 99.2% cumulative allele frequency (138 alleles) of the known HLA alleles in Japanese population at the field-2 level of resolution at six HLA loci (20 alleles for HLA-A, 37 HLA-B, 19 HLA-C, 30 HLA-DRB1, 16 HLA-DQB1, and 16 HLA-DPB1) (Table S1)

Summary

INTRODUCTION

The highly polymorphic human major histocompatibility complex (MHC), known as the human leukocyte antigen (HLA), expresses class I and II molecules (alleles) that present antigens to the T-cell receptors as part of the adaptive immune response [1,2,3,4]. A few particular HLA genes and alleles were examined in expression studies using flow cytometry and fluorolabeled monoclonal antibodies to measure the intensity of HLA protein surface expression [20, 21, 37] and by quantitative reverse transcription PCR (qRT-PCR) to estimate HLA transcription levels [38] Microarray methods, such as Affymetrix and Illumina, using oligoprobes are useful for the semiquantification of HLA gene transcripts expressed by a larger array of HLA class I and II genes [39, 40], but like flow cytometry and qRT-PCR, they do not identify the different HLA genotypes and alleles.

MATERIALS AND METHODS

Design of Sequence Capture Probes

RESULTS

DISCUSSION

CONCLUSION

ETHICS STATEMENT