An improved and validated RNA HLA class I SBT approach for obtaining full length coding sequences

Area of Science:

• Immunogenetics
• Molecular Biology

Background:

• Human Leukocyte Antigen (HLA) class I allele polymorphism is critical for immune response.
• Current typing methods often miss variations outside exons 2 and 3, limiting functional relevance assessment.
• Over 70% of HLA class I alleles are incompletely defined by sequence data.

Purpose of the Study:

• To develop and validate an improved RNA-based sequencing approach for comprehensive HLA class I typing.
• To enable routine full-length sequencing of HLA class I alleles, including those with unknown exons.
• To address the challenge of characterizing polymorphism beyond exons 2 and 3.

Main Methods:

• Improved and validated HLA sequence-based typing (SBT) using RNA templates.
• Employed single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications.
• Utilized three forward and three reverse sequencing reactions for full-length sequencing.

Main Results:

• RNA SBT of a reference panel yielded identical results to DNA SBT for locus-specific typing.
• Group-specific RNA SBT successfully determined full-length coding sequences for alleles with unknown exons, including Null and Low expressed alleles.
• The RNA SBT approach demonstrated reliability for routine full-length allele definition.

Conclusions:

• The improved RNA SBT method provides a robust solution for comprehensive HLA class I typing.
• This approach is valuable for routine clinical applications requiring full-length allele characterization.
• Enables deeper understanding of HLA polymorphism and its functional implications.