HLA allele-specific expression: Methods, disease associations, and relevance in hematopoietic stem cell transplantation.

Varying HLA allele-specific expression levels are associated with human diseases, such as graft versus host disease (GvHD) in hematopoietic stem cell transplantation (HSCT), cytotoxic T cell response and viral load in HIV infection, and the risk of Crohn's disease. Only recently, RNA-based next generation sequencing (NGS) methodologies with accompanying bioinformatics tools have emerged to quantify HLA allele-specific expression replacing the quantitative PCR (qPCR) -based methods. These novel NGS approaches enable the systematic analysis of the HLA allele-specific expression changes between individuals and between normal and disease phenotypes. Additionally, analyzing HLA allele-specific expression and allele-specific expression loss provide important information for predicting efficacies of novel immune cell therapies. Here, we review available RNA sequencing-based approaches and computational tools for NGS to quantify HLA allele-specific expression. Moreover, we explore recent studies reporting disease associations with differential HLA expression. Finally, we discuss the role of allele-specific expression in HSCT and how considering the expression quantification in recipient-donor matching could improve the outcome of HSCT.

Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes.

Identification of human leukocyte antigen (HLA) alleles from next-generation sequencing (NGS) data is challenging because of the high polymorphism and mosaic nature of HLA genes. Owing to the complex nature of HLA genes and consequent challenges in allele assignment, Oxford Nanopore Technologies' (ONT) single-molecule sequencing technology has been of great interest due to its fitness for sequencing long reads. In addition to the read length, ONT's advantages are its portability and possibility for a rapid real-time sequencing, which enables a simultaneous data analysis. Here, we describe a targeted RNA-based method for HLA typing using ONT sequencing and SeqNext-HLA SeqPilot software (JSI Medical Systems GmbH). Twelve classical HLA genes were enriched from cDNA of 50 individuals, barcoded, pooled, and sequenced in 10 MinION R9.4 SpotON flow cell runs producing over 30,000 reads per sample. Using barcoded 2D reads, SeqPilot assigned HLA alleles to two-field typing resolution or higher with the average read depth of 1750x. Sequence analysis resulted in 99-100% accuracy at low-resolution level (one-field) and in 74-100% accuracy at high-resolution level (two-field) with the expected alleles. There are still some limitations with ONT RNA sequencing, such as noisy reads, homopolymer errors, and the lack of robust algorithms, which interfere with confident allele assignment. These issues need to be inspected carefully in the future to improve the allele call rates. Nevertheless, here we show that sequencing of multiplexed cDNA amplicon libraries on ONT MinION can produce accurate high-resolution typing results of 12 classical HLA loci. For HLA research, ONT RNA sequencing is a promising method due to its capability to sequence full-length HLA transcripts. In addition to HLA genotyping, the technique could also be applied for simultaneous expression analysis.

HLA RNA Sequencing With Unique Molecular Identifiers Reveals High Allele-Specific Variability in mRNA Expression.

The HLA gene complex is the most important single genetic factor in susceptibility to most diseases with autoimmune or autoinflammatory origin and in transplantation matching. Most studies have focused on the vast allelic variation in these genes; only a few studies have explored differences in the expression levels of HLA alleles. In this study, we quantified mRNA expression levels of HLA class I and II genes from peripheral blood samples of 50 healthy individuals. The gene- and allele-specific mRNA expression was assessed using unique molecular identifiers, which enabled PCR bias removal and calculation of the number of original mRNA transcripts. We identified differences in mRNA expression between different HLA genes and alleles. Our results suggest that HLA alleles are differentially expressed and these differences in expression levels are quantifiable using RNA sequencing technology. Our method provides novel insights into HLA research, and it can be applied to quantify expression differences of HLA alleles in various tissues and to evaluate the role of this type of variation in transplantation matching and susceptibility to autoimmune diseases.

Complement Factor H Binds to Human Serum Apolipoprotein E and Mediates Complement Regulation on High Density Lipoprotein Particles.

The alternative pathway of complement is an important part of the innate immunity response against foreign particles invading the human body. To avoid damage to host cells, it needs to be efficiently down-regulated by plasma factor H (FH) as exemplified by various diseases caused by mutations in its domains 19-20 (FH19-20) and 5-7 (FH5-7). These regions are also the main interaction sites for microbial pathogens that bind host FH to evade complement attack. We previously showed that inhibition of FH binding by a recombinant FH5-7 construct impairs survival of FH binding pathogens in human blood. In this study we found that upon exposure to full blood, the addition of FH5-7 reduces survival of, surprisingly, also those microbes that are not able to bind FH. This effect was mediated by inhibition of complement regulation and subsequently enhanced neutrophil phagocytosis by FH5-7. We found that although FH5-7 does not reduce complement regulation in the actual fluid phase of plasma, it reduces regulation on HDL particles in plasma. Using affinity chromatography and mass spectrometry we revealed that FH interacts with serum apolipoprotein E (apoE) via FH5-7 domains. Furthermore, binding of FH5-7 to HDL was dependent on the concentration of apoE on the HDL particles. These findings explain why the addition of FH5-7 to plasma leads to excessive complement activation and phagocytosis of microbes in full anticoagulated blood. In conclusion, our data show how FH interacts with apoE molecules via domains 5-7 and regulates alternative pathway activation on plasma HDL particles.

Human single-chain urokinase is activated by the omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues.

Fibrinolysis is important in cell migration and tightly regulated by specific inhibitors and activators; of the latter, urokinase (uPA) associates with enhancement of cell migration. Active uPA is formed through cleavage of the single-chain uPA (scuPA). The Salmonella enterica strain 14028R cleaved human scuPA at the peptide bond Lys158-Ile159, the site cleaved also by the physiological activator human plasmin. The cleavage led to activation of scuPA, while no cleavage or activation were detected with the mutant strain 14028R lacking the omptin protease PgtE. Complementation and expression studies confirmed the role of PgtE in scuPA activation. Similar cleavage and activation of scuPA were detected with recombinant Escherichia coli expressing the omptin genes pla from Yersinia pestis, ompT and ompP from E. coli, sopA from Shigella flexneri, and leo from Legionella pneumophila. For these omptins the activation of scuPA is the only shared function so far detected. Only poor cleavage and activation of scuPA were seen with YcoA of Y. pestis and YcoB of Yersinia pseudotuberculosis that are considered to be proteolytically inactive omptin variants. Point mutations of active site residues in Pla and PgtE had different effects on the proteolysis of plasminogen and of scuPA, indicating versatility in omptin proteolysis.