High-Throughput MICA/B Genotyping of Over Two Million Samples: Workflow and Allele Frequencies

Abstract

MICA and MICB are ligands of the NKG2D receptor and thereby influence NK and T cell activity. MICA/B gene polymorphisms, expression levels and the amount of soluble MICA/B in the serum have been linked to autoimmune diseases, infections, and cancer. In hematopoietic stem cell transplantation, MICA matching between donor and patient has been correlated with reduced acute and chronic graft-vs.-host disease and improved survival. Hence, we developed an extremely cost-efficient high-throughput workflow for genotyping MICA/B for newly registered potential stem cell donors. Since mid-2017, we have genotyped over two million samples using NGS amplicon sequencing for MICA/B exons 2-5. In donors of German origin, MICA * 008 is the most common MICA allele with a frequency of 42.3%. It is followed by MICA * 002 (11.7%) and MICA * 009 (8.8%). The three most common MICB alleles are MICB * 005 (43.9%), MICB * 004 (21.7%), and MICB * 002 (18.9%). In general, MICB is less diverse than MICA and only 6 alleles, instead of 15, account for a cumulative allele frequency of 99.5%. In 0.5% of the samples we observed at least one allele of MICA or MICB which has so far not been reported to the IPD/IMGT-HLA database. By providing MICA/B typed voluntary donors, clinicians become empowered to include MICA/B into their donor selection process to further improve unrelated hematopoietic stem cell transplantation.

High‐throughput genotyping of HLA‐G, HLA‐F, MICA, and MICB and analysis of frequency distributions in healthy blood donors from Catalonia

“…Our study is also the first to determine the frequencies of MICB alleles in Spain, providing important data that will facilitate the investigation of associations between these loci and different diseases in Spanish populations. In addition, the allele frequencies observed for MICA , MICB , and MICA*011 are very similar to previous reports for Europe 34 …”

Estimation of German KIR Allele Group Haplotype Frequencies

“…Between October 2016 and April 2019, 2.6 million donors recruited by DKMS were KIR genotyped at allelic resolution by DKMS Life Science Lab in Dresden, Germany, using next generation sequencing methods (53,55,56). Genotyping comprises the HLA loci HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1, the KIR loci KIR2DL1-5, KIR3DL1-3, KIR2DS1-5, KIR3DS1, KIR2DP1, and KIR3DP1, as well as ABO (57), RhD, CCR5 (58), and MIC-A/B (59). The KIR genotyping approach delivers both allele group information and copy numbers for every KIR gene (53).…”

MICAgen Mice Recapitulate the Highly Restricted but Activation-Inducible Expression of the Paradigmatic Human NKG2D Ligand MICA

“…However, since MICA is highly polymorphic with more than 150 alleles reported to date encoding for more than 90 different MICA proteins (50,51), certain results obtained from studies of this mouse model expressing MICA * 007 may not be generally applicable to all MICA variants: these limitations may specifically relate to functional in vivo studies with regard to MICA allelic variants substantially differing from MICA * 007 in the affinity for NKG2D (such as MICA * 004) (5,52,53) or in the membrane localization and mode of release (such as MICA * 008) (54,55).…”

“…Erythrocytes were removed by gradient centrifugation with Ficoll-Paque Plus (GE Healthcare), and NK cells were isolated from splenocytes using the mouse NK cell isolation kit II (Miltenyi Biotec) according to the manufacturer's protocol. B16F10 transductants were labeled with 50 µCi of 51 Cr (PerkinElmer, Waltham, MA) for 2 h at 37 • C. After washing, 51 Cr-labeled cells were incubated for 4 h at 37 • C with purified NK cells at different effector (E) to target (T) ratios. Subsequently, supernatants were mixed with OptiPhase SuperMix scintillation mixture in an IsoPlate-96 and measured with a MicroBeta2 plate counter (all from PerkinElmer).…”