Regulation of HLA class I expression by non-coding gene variations

Abstract

The Human Leukocyte Antigen (HLA) is a critical genetic system for different outcomes after solid organ and hematopoietic cell transplantation. Its polymorphism is usually determined by molecular technologies at the DNA level. A potential role of HLA allelic expression remains under investigation in the context of the allogenic immune response between donors and recipients. In this study, we quantified the allelic expression of all three HLA class I loci (HLA-A, B and C) by RNA sequencing and conducted an analysis of expression quantitative traits loci (eQTL) to investigate whether HLA expression regulation could be associated with non-coding gene variations. HLA-B alleles exhibited the highest expression levels followed by HLA-C and HLA-A alleles. The max fold expression variation was observed for HLA-C alleles. The expression of HLA class I loci of distinct individuals demonstrated a coordinated and paired expression of both alleles of the same locus. Expression of conserved HLA-A~B~C haplotypes differed in distinct PBMC’s suggesting an individual regulated expression of both HLA class I alleles and haplotypes. Cytokines TNFα /IFNβ, which induced a very similar upregulation of HLA class I RNA and cell surface expression across alleles did not modify the individually coordinated expression at the three HLA class I loci. By identifying cis eQTLs for the HLA class I genes, we show that the non-coding eQTLs explain 29%, 13%, and 31% of the respective HLA-A, B, C expression variance in unstimulated cells, and 9%, 23%, and 50% of the variance in cytokine-stimulated cells. The eQTLs have significantly higher effect sizes in stimulated cells compared to unstimulated cells for HLA-B and HLA-C genes expression. Our data also suggest that the identified eQTLs are independent from the coding variation which defines HLA alleles and thus may be influential on intra-allele expression variability although they might not represent the causal eQTLs.

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 a number of studies using quantitative rtPCR and/or NGS, a few-fold difference in RNA expression levels between HLA-C*03:03:01 and C*04:01:01 was observed ( 10 , 14 , 15 , 20 ). In our assay, we were able to show that in K562 cells, the DNA sequence differences between the two alleles are indeed causative in driving a few-fold difference in RNA expression.…”

“…To conduct the ectopic expression assay for different HLA-C gene alleles, we prepared HLA expression plasmids as follows. We used representative genomic DNA samples of two different HLA-C alleles, HLA-C*03:03:01 and C*04:01:01 , which are reported to have significant differences in RNA levels as measured by our team and others ( 10 , 14 , 15 , 20 ), and C*03:23N , which is a null allele identified by Shimizu et al. ( 32 ).…”

“…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.…”

Two new HLA‐DQB1*03:02:01 variants with substitutions in intron 2: HLA‐DQB1*03:02:01:19 and ‐DQB1*03:02:01:21

“…Next Generation Sequencing (NGS) technologies have greatly increased the understanding of the HLA system and have elicited the better characterisation and identification of novel HLA alleles 1,2 . Polymorphic sites within non‐coding regions, such as Untranslated Regions (UTR), or introns, might play a crucial role in gene regulation and expression 3,4 . Hence, the identification of novel HLA alleles with substitutions in non‐coding regions could be relevant for further investigations.…”

“…1,2 Polymorphic sites within non-coding regions, such as Untranslated Regions (UTR), or introns, might play a crucial role in gene regulation and expression. 3,4 Hence, the identification of novel HLA alleles with substitutions in non-coding regions could be relevant for further investigations.…”