Comparison between qPCR and RNA-seq reveals challenges of quantifying HLA expression

Abstract

Human leukocyte antigen (HLA) class I and II loci are essential elements of innate and acquired immunity. Their functions include antigen presentation to T cells leading to cellular and humoral immune responses, and modulation of NK cells. Their exceptional influence on disease outcome has now been made clear by genome-wide association studies. The exons encoding the peptide-binding groove have been the main focus for determining HLA effects on disease susceptibility/pathogenesis. However, HLA expression levels have also been implicated in disease outcome, adding another dimension to the extreme diversity of HLA that impacts variability in immune responses across individuals. To estimate HLA expression, immunogenetic studies traditionally rely on quantitative PCR (qPCR). Adoption of alternative high-throughput technologies such as RNA-seq has been hampered by technical issues due to the extreme polymorphism at HLA genes. Recently, however, multiple bioinformatic methods have been developed to accurately estimate HLA expression from RNA-seq data. This opens an exciting opportunity to quantify HLA expression in large datasets but also brings questions on whether RNA-seq results are comparable to those by qPCR. In this study, we analyze three classes of expression data for HLA class I genes for a matched set of individuals: (a) RNA-seq, (b) qPCR, and (c) cell surface HLA-C expression. We observed a moderate correlation between expression estimates from qPCR and RNA-seq for HLA-A , -B, and -C (0.2 ≤ rho ≤ 0.53). We discuss technical and biological factors which need to be accounted for when comparing quantifications for different molecular phenotypes or using different techniques. Supplementary Information The online version contains supplementary material available at 10.1007/s00251-023-01296-7.

Loss of heterozygosity impacts MHC expression on the immune microenvironment in CDK12-mutated prostate cancer

“…[ 88 ] and Aguiar, V.R.C. [ 45 ] further support this correlation, emphasizing the intricate interplay between mRNA levels and surface expression of HLA-C proteins.…”

“…S1) [ 43 , 44 ]. Our rationale for classifying cases as "MHC low" or "MHC high" is based on previous research demonstrating correlations between HLA gene expression and surface protein levels [ 45 ]. For the quartile quantification, the normalized expression values of the classical genes that composed each MHC class were used (e.g., MHC-I: HLA-A , HLA-B , HLA-C ; MHC-II: HLA-DPA1 , HLA-DPB1 , HLA-DQA1 , HLA-DQB1 , HLA-DQB2 , HLA-DRA , HLA-DRB5 , HLA-DRB6 ) [ 44 ].…”

Specificity of HLA monoclonal antibodies and their use to determine HLA expression on lymphocytes and peripheral blood stem cells

“…While the measurement of individual HLA epitopes has been successfully demonstrated by several groups using allotype-specific antibodies, 23,46 there is a lack of mAbs that encompass the entire spectrum of known HLA alleles. The recently described 47 use of molecular methods such as RNA-seq and qPCR for measuring HLA would circumvent these short-comings, however, there is not a consensus on a standard technique when measuring HLA expression at the molecular level. To the best of our knowledge, this is the first paper that verifies HLA mAbs by comparing the binding reactivity of solid-phase with cell-based assays.…”

Nucleotide alterations in the HLA-C class I gene can cause aberrant splicing and marked changes in RNA levels in a polymorphic context-dependent manner

“…In this study, we developed a novel ectopic expression assay to investigate the impact of HLA polymorphisms on RNA expression across the entire gene context for the class I HLA-C gene focusing mainly on three different alleles. First, we validated the assay by examining differential RNA expression levels for two HLA alleles, HLA-C*03:03:01 and C*04:01:01 , which showed significantly different levels of RNA expression in RNA-seq association studies conducted by our team and others ( 10 , 14 , 15 , 20 ). Subsequently, we employed the assay to examine the influence of a polymorphism that was implicated to a null phenotype of the HLA-C*03:23N at single nucleotide resolution, considering different polymorphic contexts of the gene.…”