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.

Keywords: MICA; MICB; NGS; allele; genotyping; hematopoietic stem cell transplantation; high-throughput; next generation sequencing.

Figure 1

Primer locations and PCR amplification products for exons 2–5 of MICA/B. Primers (arrows) bind to both MICA and MICB and generate three amplicons per gene in one PCR reaction. Product lengths are between 417 and 480 bp. Note that not all bases of exons 4 and 5 are covered.

Figure 2

Allele frequencies of MICA. First-field-resolution allele frequencies are based on 1,201,896 samples from donors of German descent. Alleles contributing to a cumulative allele frequency of 99.5% are shown against a colored background and allele frequencies below 0.003 are additionally plotted in an inlay. If ambiguities exist, allele groups are used (#) and the ambiguity is described in Table 1.

Figure 3

Allele frequencies of MICB. First-field-resolution allele frequencies are based on 1,201,896 samples from donors of German descent. Alleles contributing to a cumulative allele frequency of 99.5% are shown against a colored background and allele frequencies below 0.004 are additionally plotted in an inlay. If ambiguities exist, allele groups are used (#) and the ambiguity is described in Table 1.