A Typing System for the Major Histocompatibility Complex Class I Chain Related Genes A and B Using Polymerase Chain Reaction with Sequence-Specific Primers

Abstract

The Major Histocompatibility Complex (MHC) class I chain related (MIC) A and B genes are important additional loci within the MHC. We have developed a MICA and MICB typing system using the polymerase chain reaction with sequence-specific primers (PCR-SSP), which operates under the same conditions as our routine HLA-A, -B, and -C typing method. We designed 95 primers in 84 SSP mixtures for MICA and 39 primers in 29 mixtures for MICB. This detected and differentiated all 55 MICA and 19 MICB alleles (except MICA*00701 from MICA*026, MICA*00201 from MICA*020, and three MICB alleles, which are intronic variations). A computer program confirmed the MICA amplification reactivity of each SSP mixture and evaluated the typing set for MICA allele combination ambiguities. Seventy-six "reference" DNA samples were used for validation: 50 from International Histocompatibility Workshop B lymphoblastoid cell lines (IHW BCLs) and 26 MICA-typed samples from two laboratories. The reference material identified 28 out of the 55 MICA alleles and 13 of the 19 MICB alleles, and directly validated 62 of the 84 MICA and 20 of the 29 MICB SSP mixtures. Our genotyping agreed with 283 out of the 286 (98.95%) MICA and MICB reference laboratories' allele assignments or the consensus assignments. Two of the discrepancies remain unresolved, whereas one was probably due to a reference laboratory's failure to differentiate alleles differing in exon 5 of the MICA gene. A comparison of the MICA and MICB allele assignments between laboratories identified a "disagreement rate" of 19.4% for MICA alleles and 13.1% for MICB alleles. Accordingly, we have compiled "consensus" MICA and MICB genotypes for the 50 IHW BCLs tested, which have been confirmed by our typing. We also typed 166 random blood donors. Their MICA and MICB carriage and allele frequencies and HLA-B, MICA, MICB linkage disequilibrium parameters and haplotype frequencies largely concurred with other published data on United Kingdom subjects, further supporting the validity of our typing system. This PCR-SSP system is a simple, reliable and rapid technique for typing MICA and MICB alleles. It is easily updated as new alleles are identified but clearly requires a continuing validation review until all known MICA and MICB alleles have been identified.

Conserved extended haplotypes of the major histocompatibility complex: further characterization

“…68 Likewise, we confirmed the MICA type of the cell line HOR as *004 (but not as *007). 63 The cell line CF996 was typed as MICA*019 by us in agreement with some others 68,71 but not with all. 65,66 The MICA type of cell line HO301 was *011 but not *008.…”

“…73 We investigated the relationship of this polymorphism with HLA haplotypes. With one exception (CEH 58.1), the minor allele þ 130A was on the LTA- 70 Rees et al 71 recently updated the MICA (and MICB) typings that were inconsistent in the literature using a PCR with sequence-specific primers (PCR-SSP) they developed. Most importantly, the cell line EHM -the only reference cell line for MICA*028 -was retyped as heterozygote for MICA alleles *00201/*020 and *00901.…”

“…These results are presented in Supplementary Table. One noteworthy discrepancy concerns the cell line LUY. This cell line has been typed as MICA*049 71 or as *00901, 66,68,69 and listed as heterozygote for *00901 and *049 in the IHWG MICA Panel and IMGT/HLA MICA Sequence Database. Our SBT method typed this cell line as homozygous for MICA*00901 (and STR allele A6).…”

High frequencies of alleles MICA*020 and MICA*027 in Amerindians and evidence of positive selection on exon 3

“…21,40 Surprisingly, it seems that all populations have high frequencies of MICA alleles that may have an altered function or no function at all. If we sum the frequencies (Table 1) of the deletion and of alleles MICA*00801, MICA*00802, MICA*00804, MICA*023, MICA*028, MICA*053 (alleles with the G insertion in the STR of exon 5), MICA*010, MICA*025, MICA*054 (alleles that have no surface expression because of a proline-forarginine substitution in the a1 domain), MICA*015 and MICA*017 (alleles with a G deletion at the end of exon 4, encoding a polypeptide with a different amino-acid sequence in the transmembrane region and with a short cytoplasmic tail), the total frequency is as high as 50-63% in Euro-Americans 32,33 and Welsh 31 and varies from 30 to 44% in other non-Amerindian populations. In Amerindians, this summed frequency is approximately 18% in Wichi, Terena 27 and Guarani Kaiowá and 30-40% in the other populations.…”

“…Assignment of each individual's genotype was achieved through the software HLA Visual 2.2 (One Lambda Inc.). Ambiguous results were solved by PCR-SSP (PCR-sequence-specific primers) as described by Collins et al 45 and Rees et al 31 The ambiguity between the alleles MICA*00901 and MICA*049 was solved by PCR-RFLP (PCR-restriction fragment length polymorphism), amplifying the region containing the SNP that differs between the two alleles with the primers MICAF: 5 0 -agctcgtgagcctgca-3 0 and MICAR: 5 0 -agtggagccagtggacccaag-3 0 and then digesting the products with BseGI restriction enzyme.…”