Allele-Level KIR Genotyping of More Than a Million Samples: Workflow, Algorithm, and Observations

Area of Science:

• Immunogenetics
• Molecular Biology
• Genomics

Background:

• Killer-cell immunoglobulin-like receptor (KIR) genes are crucial for natural killer cell function and impact immune-mediated diseases and hematopoietic stem cell transplantation (HSCT).
• The genetic complexity of the KIR locus, including extensive copy number variation (CNV) and allelic diversity, has limited high-resolution characterization to small cohorts.
• Accurate KIR genotyping is essential for understanding immune responses and optimizing transplantation protocols.

Purpose of the Study:

• To develop and validate a comprehensive, high-throughput, next-generation sequencing-based approach for high-resolution KIR genotyping.
• To enable precise determination of both KIR gene copy numbers and allelic variations across large populations.
• To facilitate the application of KIR genotyping in large-scale studies, including stem cell donor registries and population genetics.

Main Methods:

• Utilized next-generation sequencing with PCR amplification of specific KIR exons.
• Developed a workflow to simultaneously determine KIR gene copy number and allelic information.
• Validated the approach through replicate analysis (n=190) and independent sample genotyping (n=360).

Main Results:

• Achieved high precision (>99.9%) and accuracy (>99%) in KIR genotyping.
• Successfully genotyped 1.8 million stem cell donor registry samples, revealing extensive KIR allele diversity.
• Identified over 2,000 novel KIR variants, highlighting the significant unexplored genetic diversity in the KIR region.

Conclusions:

• The developed cost-efficient, high-resolution KIR genotyping method is robust and scalable.
• This approach can be routinely applied to stem cell donor registries to improve unrelated donor HSCT outcomes by incorporating KIR as a selection criterion.
• The method is suitable for various research applications, including population genetics and disease association studies requiring detailed KIR characterization.