CONCLUSION

1. Re-examination and Completion of MICA Typings on Conserved Extended Haplotypes: Relationship with Other Polymorphisms M Tevfik Dorak 1, Wenshuo Shao 2, Helmut KG Machulla 3, Myoung Hee Park 4, Elena Lobashevsky 2, Jianming Tang 2, Richard Kaslow 2 1 School of Clinical Medical Sciences, Newcastle University, U.K; 2 Dept of Epidemiology, University of Alabama at Birmingham, USA; 3 Interbranch HLA Lab, Martin Luther University Medical School, Germany; 4 Dept of Lab Medicine, Seoul National University College of Medicine, Korea INTRODUCTION Despite having numerous published reports on the MHC loci in conserved extended haplotypes (CEH), also called ancestral haplotypes, considerable amount of data are still missing and there are also inconsistencies. We aimed to unambiguously characterise 37 CEHs at a number of MHC loci, including MICA, using primarily IHWG reference cell lines in a single study (Dorak et al, 2006). Here, we present the MICA results. RESULTS Table 1 summarises the complete results on CEHs. We obtained the MICA types for CEHs 35.3, 35.4, 44.4, 50.1, 51.1, 62.2, 64.1 and 65.2 (represented by cell lines SPE/G, TISI, HOR, EAV/AC (heterozygote), BM92, COL/E, CF996 and HO301). The whole cell panel did not have any example of MICA alleles *005, *013, and *014 similar to the results of other studies. We confirmed that all CEHs with the same HLA-B allele have the same MICA STR allele except HOR. The cell line HOR is a heterozygous example of CEH 44.4 but differs from other B*4403-bearing CEHs (STR allele A4 as opposed to A6). One observation was that different alleles of serologically indistinguishable specificities (*1401/1402; *3501/3502; *4001/4002; *4402/4403) were associated with different MICA STR alleles in CEHs. MICA*00801 (which includes STR A5.1 in its allele definition) was present on a highly divergent set of CEHs: 7.1; 7.2; 8.1; 13.1; 37.1; 44.1; 47.1; 60.1; 60.2 and 60.3. This set did not share any other allele at other loci. Table 1. MICA typings in 37 conserved extended haplotypes A SNP at nt130 of exon 3 of the MICA gene has functional importance in binding to NKG2D receptor of NK cells. We investigated the relationship of this polymorphism with HLA haplotypes. With one exception (CEH 58.1), the minor allele +130A was on the LTA +252A - NFKBIL1 -62A haplotype (CEH 18.1, 18.2, 18.3, 35.2, 35.3, 38.1, 51.1, 54.1, 57.1, 65.1 and 65.2). 10 out of 11 haplotypes that carried LTA allele +252G had MICA +130G. This relationship may confound associations with either loci. MATERIAL AND METHODS We genotyped 101 IHWG reference cell lines and nine additional anonymous samples representing all 37 unambiguously characterized CEHs at MICA, NFKBIL1, LTA, NCR3, AIF1, HSPA1A, HSPA1B, BF, NOTCH4 and a SNP at HLA-DQA1 as well as MICA, NOTCH4, HSPA1B and all five TNF STR polymorphisms (Dorak et al, 2006). Complete typing results on these samples are available on request as an Excel file. CEH assignments were taken from the latest update (Cattley et al, 2000) and reviews (Dawkins et al, 1999; Yunis, 2003). Five CEHs (35.1; 58.2; 59.1; 61.1; 62.4) usually included in provisional lists of CEHs could not be analysed because of insufficient characterisation of them, in particular at the DNA level. Ten additional HLA-B - DRB1 homozygous samples from normal populations were also included in the study. Three of these samples were from our previous newborn study (origin: South Wales, U.K.) homozygous for B*0801-DRB1*0301, B*1302-DRB1*0701 and B*4402-DRB1*0401, respectively; two samples from the Korea Marrow Donor Program (B*3701-DRB1*1001); two from a Mongolian population study (B*5801- DRB1*0301); and two samples a Zambian HIV-1 transmission study (B*4201-DRB1*0302). We only typed the cell lines that were representatives of CEHs with missing data or had inconsistent MICA results in the literature using the modified SBT scheme described by us (Shao et al, 2004). CONCLUSION The overall CEH study 1) provided an extensive catalogue of MHC polymorphisms in all CEHs; 2) unravelled interrelationships between HLA and non-HLA haplotypical lineages; 3) resolved reported typing ambiguities, and 4) described haplospecific markers for a number of CEHs. The complete results on multiple MHC loci on CEHs will be useful in design and interpretation of HLA and disease association studies. This study highlighted the need to reexamine previously reported genotype data. This is most urgent for the complotypes, the main characteristics of CEHs, which are not routinely typed by DNA-based methods. Rees et al (2005) updated the MICA/B typings that were inconsistent in the literature using a new PCR-SSP scheme. The cell line EHM -the only reference cell line for MICA*028- was typed as heterozygote for MICA*00201/020 and *00901. We obtained the same result by SBT. The cell line TISI was reported as MICA*017 elsewhere but our result agreed with that of another study (*016). We confirmed the MICA type of the cell line HOR as *004 (but not as *007). The cell line CF996 was typed as MICA*019 by us disagreeing with some other results. The MICA type of cell line HO301 was *011 but not *008. The cell line LUY has been reported to be MICA*049 or *00901, and listed as *00901 / *049 in the IHWG and IMGT-HLA databases. Our SBT method typed this cell line as homozygous for MICA*00901 (and STR allele A6). The only difference between MICA*00901 and *049 is at the nucleotide position 109 in exon 3 and LUY has G at this position (not C). REFERENCES Cattley SK, Williamson JF, Tay GK, Martinez OP, Gaudieri S, Dawkins RL (2000) Further characterization of MHC haplotypes demonstrates conservation telomeric of HLA-A: update of the 4AOH and 10IHW cell panels. Eur J Immunogenet 27:397-426 Dawkins R, Leelayuwat C, Gaudieri S et al (1999) Genomics of the MHC: haplotypes, duplication, retroviruses and disease. Immunol Rev 167:275-304 Dorak MT, Shao W, Machulla HKG, Lobashevsky ES, Tang J, Park MH, Kaslow, RA (2006) Conserved Extended Haplotypes of the Major Histocompatibility Complex: Further Characterisation. Genes Immun (in press) Rees MT, Downing J, Darke C (2005) A typing system for the MHC class I chain related genes A and B using PCR with sequence-specific primers. Genet Test 9:93-110 Shao W, Lobashevsky ES, Kaslow RA, Dorak MT (2004) MICA intron 1 sequences of conserved extended HLA haplotypes: implications for sequencing-based typing. Genes Immun 5:371-4 Yunis EJ, Larsen CE, Fernandez-Vina M et al (2003) Inheritable variable sizes of DNA stretches in the human MHC: conserved extended haplotypes and their fragments or blocks. Tissue Antigens 62:1-20 Several studies reported a variant form of the MICA-STR allele R6 on different examples of CEH 65.1. We were unable to detect any difference between the cell lines representing CEH 65.1 and other cell lines carrying R6. The reason may be that we separated amplification products on polyacrylamide gels rather than with capillary electrophoresis. We therefore could not confirm the presence of a variant MICA-STR allele. ONLINE DATABASES FOR CELL LINE DATA European Collection of Cell Cultures Centre (ECACC): http://www.ecacc.org.uk International Histocompatibility Working Group (IHWG) Cell & Gene Bank: http://www.ihwg.org/shared/cbankover.htm International Immunogenetics Project (IMGT) HLA Sequence Database: http://www.ebi.ac.uk/cgi-bin/imgt/hla/get_all.cgi