Design and Validation of a Multiplex KIR and HLA Class I Genotyping Method Using Next Generation Sequencing

Area of Science:

• Immunogenetics
• Molecular Biology
• Next-Generation Sequencing

Background:

• Killer cell immunoglobulin-like receptors (KIR) are highly polymorphic regulators of natural killer (NK) cells, crucial for hematopoietic stem cell transplantation (HSCT).
• Current KIR genotyping methods, such as PCR-SSO and PCR-SSP, lack the resolution needed for optimal donor selection in HSCT.
• Accurate KIR and HLA genotyping is vital for achieving graft-versus-leukemia effects and improving HSCT success rates.

Purpose of the Study:

• To develop a novel multiplex, long-range PCR strategy for simultaneous, high-resolution KIR and HLA class I genotyping.
• To enable efficient and cost-effective KIR and HLA typing using next-generation sequencing (NGS).
• To validate the reliability and accuracy of the new NGS-based method compared to existing techniques.

Main Methods:

• A multiplex, long-range PCR protocol was designed to amplify all 14 KIR genes, 2 KIR pseudogenes, and HLA class I genes.
• Amplified DNA was sequenced using an Illumina platform, followed by bioinformatics analysis for genotyping.
• KIR genotyping utilized virtual hybridization of gene-specific probes, while HLA genotyping employed specialized software (GenDx NGSengine).

Main Results:

• The NGS-based method achieved 100% concordance with previous PCR-SSO characterization for both KIR and HLA assignments in 192 DNA samples.
• The protocol successfully amplified and allowed for the detection of the presence or absence of specific KIR genes.
• The method demonstrated high reliability in distinguishing between the presence and absence of KIR genes based on virtual probe detection.

Conclusions:

• The developed multiplex PCR NGS-based strategy offers an efficient and potentially less costly method for KIR-ligand genotyping.
• This approach provides accurate gene presence/absence determination, crucial for donor selection in HSCT.
• Future development of KIR-specific software could enable allele-level resolution, further enhancing its utility.