Aim: Next to KIR, located on the leukocyte receptor complex on chromosome 19, is the LILR/LAIR gene cluster. Understanding the complex interplay between LILR/LAIR, HLA molecules, and immune responses holds promise for refining transplantation strategies and enhancing patient outcomes post-HSCT. Variations in LILR/LAIR gene copy numbers and sequence polymorphisms may impact the functional repertoire and their role in immune modulation and HSC matching between donor and recipients. We set out to develop a LILR/LAIR gene capture and sequencing method to be able to analyze this variation.
Methods: Hybrid capture probes were designed for the enrichment of the Leukocyte Receptor Complex (LRC) region containing the LILR/LAIR genes, followed by subsequent long read sequencing of the obtained libraries on a GridION (ONT). An experimental version of NGSengine-Turbo software (GenDx) was equipped with a newly developed reference library for analysis of LILR/LAIR genes using reference sequences obtained from GenBank and ENSEMBL, as well as new sequences derived from 8 cell lines (Coriell) as determined by GenDx.
Results: We obtained sequence data for all the functional LILR/LAIR genes and 2 LILR pseudogenes. Sequence reads for the following genes were detected: LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3 LILRB4, LILRB5, LILRP1, LILRP2, LAIR1 and LAIR2. The reads covered all exons for these genes and for most genes whole gene coverage (including introns) was achieved. Several challenges were encountered during analysis of the LILR/LAIR genes. Some of the genes contain very large introns (e.g. LILRA2 over 10kb). Moreover, some alignment issues were apparent due to the fact that some genes are located very close together and therefore can be found on the same long sequence reads. The current lack of a nomenclature system for the LILR/LAIR alleles was resolved by establishing a new nomenclature, similar to that used for the neighboring KIR locus. Interestingly, in the first small panel of 8 samples, multiple polymorphic sequences were discovered, representing new intron variants as well as a few exon variants.
Conclusion: Hybrid capture was successfully applied using ONT-based long read sequencing methods to type LILR/LAIR genes. We obtained whole gene sequence data for most, and full exon data for all, LILR/LAIR genes. In addition, we present a suggestion for a nomenclature system for the newly discovered alleles.
