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

Area of Science:

• Immunogenetics
• Molecular Biology
• Genomics

Background:

• The human major histocompatibility complex (MHC), or human leukocyte antigen (HLA), is highly polymorphic with over 25,000 alleles.
• HLA diversity influences transplant outcomes, infection susceptibility, and autoimmune diseases.
• Next-generation sequencing (NGS) offers new ways to study HLA diversity's impact on allele expression.

Purpose of the Study:

• To describe a novel NGS capture RNA-Seq method for genotyping all 12 classical HLA loci.
• To assess allelic imbalance by quantifying HLA allele RNA levels.
• To evaluate the method's application in healthy donor samples.

Main Methods:

• Target enrichment using RNA-specific HLA biotinylated oligonucleotide capture probes.
• Conversion of total RNA to a sequencing-ready complementary DNA (cDNA) library.
• Application of the method to peripheral blood mononuclear cells and umbilical cord blood cells.

Main Results:

• Successful genotyping of all 12 classical HLA loci.
• Quantification of allele RNA levels and assessment of allelic imbalance.
• Demonstration of significant differential allelic expression in 10 HLA loci (P < 2.1 × 10^-15).
• Corroboration of results by independent methods.

Conclusions:

• The developed NGS capture RNA-Seq method accurately genotypes HLA loci and measures allele expression.
• Significant differential allelic expression exists across multiple HLA loci.
• This method has broad applications in understanding graft rejection and HLA-related diseases.